WNT surrogate molecules and uses thereof

ABSTRACT

The present invention provides Wnt pathway agonists and related compositions, which may be used in any of a variety of therapeutic methods for the treatment of diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application under 35 U.S.C. § 371 of International Application No. PCT/US2018/066616, filed Dec. 19, 2018, which claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/607,875, filed Dec. 19, 2017, U.S. Provisional Application No. 62/641,217, filed Mar. 9, 2018, and U.S. Provisional Application No. 62/680,522, filed Jun. 4, 2018, each of which is incorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is SRZN_006_03WO_ST25.txt. The text file is 1.2 MB, was created on Dec. 19, 2018, and is being submitted electronically via EFS-Web.

BACKGROUND Technical Field

The present invention relates generally to Wnt signaling pathway agonist molecules, compositions, and methods of using the same. Such molecules are useful, for example, in modulating Wnt signaling pathways.

Description of the Related Art

Wnt (“Wingless-related integration site” or “Wingless and Int-1” or “Wingless-Int”) ligands and their signals play key roles in the control of development, homeostasis and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste bud, ovary, cochlea and many other tissues (reviewed, e.g., by Clevers, Loh, and Nusse, 2014; 346:1248012). Modulation of Wnt signaling pathways has potential for treatment of degenerative diseases and tissue injuries.

One of the challenges for modulating Wnt signaling as a therapeutic is the existence of multiple Wnt ligands and Wnt receptors, Frizzled 1-10 (Fzd1-10), with many tissues expressing multiple and overlapping Fzds. Canonical Wnt signals also involve Low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors, which are broadly expressed in various tissues, in addition to Fzds. Accordingly, there is clearly a need in the art for binding moieties that specifically bind to one or more Fzd, LRP5, or LRP6 to modulate Wnt signaling pathways. The present invention addresses this need.

BRIEF SUMMARY

In various embodiments, the present invention provides WNT surrogate molecules and related uses thereof.

In one embodiment, the disclosure provides a soluble, bivalent, bispecific Wnt surrogate molecule, wherein the Wnt surrogate molecule comprises: (i) one or more regions that specifically binds to one or more Frizzled (Fzd) receptor (a Fzd binding region); and (ii) one or more regions that specifically binds to a Low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and/or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) (a LRP5/6 binding region).

In particular embodiments, the Wnt surrogate molecule comprises two or more Fzd binding regions and two or more LRP5/6 binding regions. In particular embodiments, one or more Fzd binding regions comprise one or more antigen-binding fragments of an antibody. In particular embodiments, one or more antigen-binding fragments are selected from the group consisting of: IgG, scFv, Fab, and VHH or sdAb

In particular embodiments, any of the Fzd antigen-binding fragments comprise: (i) CDRH1, CDRH2 and CDRH3 sequences set forth for any of the antibodies of Tables 1A or 1B; and/or (ii) CDRL1, CDRL2 and CDRL3 sequences set forth for any of the antibodies of Tables 1A or 1B, or a variant of said Fzd binding region comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid substitutions in said CDR sequences. In particular embodiments, any of the Fzd binding regions comprise an amino acid sequence having at least 90% identity to any of the sequences set forth in SEQ ID NOs:1-65 or 129-132, or an antigen-binding fragment thereof.

In particular embodiments, any of the Fzd binding regions bind to one or more of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzle 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). In particular embodiments, any of the Fzd binding region binds to two or more of Frizzled 1 (Fzd1), Frizzled 2 (Fzd2), Frizzled 3 (Fzd3), Frizzled 4 (Fzd4), Frizzled 5 (Fzd5), Frizzled 6 (Fzd6), Frizzled 7 (Fzd7), Frizzled 8 (Fzd8), Frizzled 9 (Fzd9), and Frizzled 10 (Fzd10). In particular embodiments, any of the Fzd binding region binds to: (i) Fzd1, Fzd2, Fzd7 and Fzd9; (ii) Fzd1, Fzd2 and Fzd7; (iii) Fzd5 and Fzd8; (iv) Fzd5, Fzd7 and Fzd8; (v) Fzd1, Fzd4, Fzd5 and Fzd8; (vi) Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8; (vii) Fzd4 and Fzd9; (viii) Fzd9 and Fzd10; (ix) Fzd5, Fzd8 and Fzd10; or (x) Fzd4, Fzd5 and Fzd8; Fzd1, Fzd5, Fzd7 and Fzd8.

In particular embodiments, any of the surrogate molecules comprise one or more LRP5/6 binding regions comprise one or more antigen-binding fragments of an antibody. In particular embodiments, the one or more antigen-binding fragments are selected from the group consisting of: IgG, scFv, Fab, and VHH or sdAb In particular embodiments, any of the one or more LRP5/6 binding regions or antigen-binding fragments comprise: (i) CDRH1, CDRH2 and CDRH3 sequences set forth for any of the antibodies of Table 2; and/or (ii) CDRL1, CDRL2 and CDRL3 sequences set forth for any of the antibodies of Table 2, or a variant of said LRP5/6 binding region comprising one or more amino acid modifications, wherein said variant comprises less than 8 amino acid substitutions in said CDR sequences. In particular embodiments, any of the one or more LRP5/6 binding regions comprise an amino acid sequence having at least 90% identity to any of the sequences set forth in SEQ ID NOs:66-88 or 133, or an antigen-binding fragment thereof.

In particular embodiments of any of the Wnt surrogate molecules, the Fzd binding region comprising a Fab, and the LRP5/6 binding region comprising a VHH or sdAb In particular embodiments, the Fab is present within a full immunoglobulin (Ig), optionally an IgG, comprising a light chain and a heavy chain. In particular embodiments, the LRP5/6 binding region is fused to the N-terminus or the C-terminus of the heavy chain. In particular embodiments, the LRP5/6 binding region is fused to the N-terminus or the C-terminus of the light chain. In certain embodiments, the LRP5/6 binding region is fused to the N-terminus of the heavy chain of the full Ig or the N-terminus of the light chain of the full Ig. In certain embodiments, the LRP5/6 binding region is fused to the C-terminus of the heavy chain of the full Ig or the C-terminus of the light chain of the full Ig. In certain embodiments, the variable light chain region of the LRP5/6 binding Fab is fused to the N-terminus of the variable heavy chain region of the full Ig. In certain embodiments, the variable light chain region of the LRP5/6 binding Fab is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable heavy chain region of the LRP5/6 binding Fab is fused to the N-terminus of the variable light chain region of the full IgG. In particular embodiments, any of the LRP5/6 binding region is fused to the heavy chain or the light chain via one or more linker moiety.

In certain embodiments of any of the Wnt surrogate molecules, the Fzd binding region comprises a VHH or sdAb and the LRP5/6 binding region comprises a Fab. In particular embodiments, the Fab is present within a full immunoglobulin (Ig), optionally an IgG, comprising a light chain and a heavy chain. In certain embodiments, the Fzd binding region is fused to the N-terminus or the C-terminus of the heavy chain. In certain embodiments, the Fzd binding region is fused to the N-terminus or the C-terminus of the light chain. In some embodiment, the Fzd binding region is fused to the N-terminus of the heavy chain of the full Ig or the N-terminus of the light chain of the full Ig. In some embodiments, the Fzd binding region is fused to the C-terminus of the heavy chain of the full Ig or the C-terminus of the light chain of the full Ig. In some embodiments, the variable light chain region of the Fzd binding Fab is fused to the N-terminus of the variable heavy chain region of the full Ig. In some embodiments, the variable light chain region of the Fzd binding Fab is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable heavy chain region of the Fzd binding Fab is fused to the N-terminus of the variable light chain region of the full IgG. In certain embodiments, any of the Fzd binding region is fused to the heavy chain or the light chain via one or more linker moiety.

In another embodiment, any of the Fzd binding region comprises a Fab or Fv, and the LRP5/6 binding region comprises a Fab or Fv. In particular embodiments, the Fab of the Fzd binding region or the Fab or Fv of the LRP5/6 binding region is present within a full immunoglobulin (Ig), optionally an IgG, comprising a light chain and a heavy chain. In particular embodiments, only one of the Fab of the Fzd binding region or the Fab of the LRPp5/6 binding region is present within the full immunoglobulin (Ig). In particular embodiments, the Fab or Fv of the Fzd binding region is present within the full Ig. In particular embodiments, the Fab or Fv of the LRP5/6 binding region is fused to the N-terminus of the Ig. In particular embodiments, the Fab of the LRP5/6 binding region is fused to the C-terminus of the Ig. In a further embodiment, the Fab of the LRP5/6 binding region is present within the full Ig. In particular embodiments, the Fab of the Fzd binding region is fused to the N-terminus of the Ig. In particular embodiments, the Fab of the Fzd binding region is fused to the C-terminus of the Ig. In some embodiments, the variable light chain region of the Fzd binding Fab is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable heavy chain region of the Fzd binding Fab is fused to the N-terminus of the variable light chain region of the full IgG. In some embodiments, the variable light chain region of the LRP5/6 binding Fv is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable heavy chain region of the LRP5/6 binding Fv is fused to the N-terminus of the variable light chain region of the full IgG. In some embodiments, the variable heavy chain region of the LRP5/6 binding Fv is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable light chain region of the LRP5/6 binding Fv is fused to the N-terminus of the variable light chain region of the full IgG. In some embodiments, variable light chain region of the Fzd binding Fv is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable heavy chain region of the Fzd binding Fv is fused to the N-terminus of the variable light chain region of the full IgG. In some embodiments, the variable heavy chain region of the Fzd binding Fv is fused to the N-terminus of the variable heavy chain region of the full Ig, and the variable light chain region of the Fzd binding Fv is fused to the N-terminus of the variable light chain region of the full IgG.

In another embodiment, any of the Fzd binding regions comprises a VHH or sdAb, and the LRP5/6 binding region comprises a VHH or sdAb. In particular embodiments, the Fzd binding region is fused to the Lrp5/6 binding region, and wherein the Fzd binding region or the LRP5/6 binding region is fused to an Fc region. In particular embodiments, the Fzd binding region is fused to the N-terminus of an Fc region, and wherein the LRP5/6 binding region is fused to the C-terminus of an Fc region. In particular embodiments, the Fzd binding region is fused to the C-terminus of an Fc region, and wherein the LRP5/6 binding region is fused to the N-terminus of an Fc region.

In a further embodiment, any of the antibodies or one or more antigen-binding fragment thereof, is humanized. In a further embodiment, any of the Wnt surrogate molecules binds to one or more Fzd receptor with a K_(D) of 50 μM or lower. In further embodiments, any of the Wnt surrogate molecules binds to LRP5 and/or LRP6 with a K_(D) of 50 μM or lower.

In a further embodiment, any of the Wnt surrogate molecule modulates a Wnt signaling pathway in a cell, optionally a mammalian cell. In particular embodiments, the Wnt surrogate molecule increases signaling via the Wnt signaling pathway in the cell. In particular embodiments, the Wnt signaling pathway is a canonical Wnt signaling pathway or a non-canonical Wnt signaling pathway.

In a related embodiment, the present disclosure provides an isolated polynucleotide encoding a polypeptide sequence comprising one or more of the Fzd binding regions and/or one or more of the LRP5/6 binding regions of a Wnt surrogate molecule. In particular embodiments, the present disclosure provides an expression vector comprising the isolated polynucleotide. In further particular embodiments, the present disclosure provides an isolated host cell comprising the expression vector.

In a related embodiment, the present disclosure provides a pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier, and a therapeutically effective amount of any of the Wnt surrogate molecules disclosed herein.

In related embodiments, the present disclosure provides a method for agonizing a Wnt signaling pathway in a cell, comprising contacting the cell any of the Wnt surrogate molecules, wherein the Wnt surrogate molecule is an agonist of a Wnt signaling pathway.

In particular embodiments, the present disclosure provides a method for treating a subject having a disease or disorder associated with reduced Wnt signaling, comprising administering to the subject an effective amount of the pharmaceutical composition, wherein the Wnt surrogate molecule is an agonist of a Wnt signaling pathway. In particular embodiments, the disease or disorder is selected from the group consisting of: bone fractures, osteoporosis (e.g., post-menopausal osteoporosis), osteoporotic fractures, spinal fusion, vertebral compression fracture, pre-operative spinal surgery optimization, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implantation, periodontal diseases, maxillofacial reconstruction, osteonecrosis of the jaw, osteoarthritis (OA), muscular dystrophy, muscle atrophy resulting from sarcopenia or cachexia, alopecia, hearing loss, including regeneration of inner and outer auditory hair cells, vestibular hypofunction, macular degeneration, vitreoretinopathy, diseases of retinal degeneration, including diabetic retinopathy, diseases/disorders affecting the integrity of the blood brain barrier, Fuchs' dystrophy, stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, spinal cord injuries, oral mucositis, short bowel syndrome, inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), in particular IBD with fistula formation, metabolic syndrome, diabetes, dyslipidemia, pancreatitis, exocrine pancreatic insufficiency, wound healing, diabetic foot ulcers, coronary artery disease, acute kidney injuries, chronic kidney diseases, chronic obstructive pulmonary diseases (COPD), pulmonary fibrosis, including idiopathic pulmonary fibrosis, acute liver failure, acute alcoholic liver injuries, chronic liver diseases with hepatitis C virus (HCV), HCV subjects post-antiviral drug therapies, chronic liver diseases with hepatitis B virus (HBV), fibrosis, HBV subjects post-antiviral drug therapies, chronic alcoholic liver diseases, alcoholic hepatitis, non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis (NASH), cirrhosis, and chronic liver insufficiencies of all causes. In certain embodiments, the disease or disorder is a bone disease or disorder. In particular embodiments, the disease or disorder is a bone disease or disorder, and the Wnt surrogate molecule binds Fzd1, Fzd2, and FZD7, and binds LRP5 and/or LRP6. In certain embodiments, the disease or disorder is a bone disease or disorder, and the Wnt surrogate molecule binds Fzd1, Fzd2, FZD7, Fzd5 and Fzd8, and also binds LRP5 and/or LRP6.

In another related embodiment, the present disclosure provides a method for increasing bone mineral density, increasing bone volume, increasing bone cortical thickness, increasing bone mineral apposition rate, increasing bone stiffness, increasing bone biomechanical strength, increasing resistance to bone fracture, decreasing bone resorption, or decreasing bone loss associated with osteoporosis, in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a Wnt surrogate molecule, wherein the Wnt surrogate molecule is an agonist of a Wnt signaling pathway. In certain embodiments, the Wnt surrogate molecule binds Fzd1, Fzd2, and FZD7, and binds LRP5 and/or LRP6. In certain embodiments, the Wnt surrogate molecule binds Fzd1, Fzd2, FZD7, Fzd5 and Fzd8, and also binds LRP5 and/or LRP6.

In particular embodiments, methods of the invention, including those related to treating or preventing a bone disease or disorder, such as osteoporosis (e.g., post-menopausal osteoporosis), further comprise providing the subject an antiresorptive agent (in combination with a Wnt surrogate molecule). Examples of anti-resorptive agents include, but are not limited to, bisphosphonates or selective estrogen receptor modulators. Antiresorptive agents are used to increase bone strength in individuals with osteoporosis and include five principal classes of agents: bisphosphonates, estrogens, selective estrogen receptor modulators (SERMs), calcitonin and monoclonal antibodies such as denosumab, any of which may be used. Illustrative examples of antiresorptive agents include, but are not limited to: bisphosphonates, e.g., alendronate-generic medication (Brand name: Fosamax™, Fosamax™ Plus D), risedronate (Brand name: Actonel™, Actonel™ with Calcium), ibandronate (Brand name: Boniva™), and zoledronic acid (Brand name: Reclast™); other antiresorptives, e.g., estrogen therapy or hormone therapy, raloxifene (Brand name: Evista™), and denosumab (Prolial™); and anabolic medication, e.g., teriparatide (Forteo™).

In a further related embodiment, the present disclosure provides a method for increasing liver to body weight ratio, promoting liver regeneration, increasing liver cell proliferation or mitosis, decreasing liver fibrosis, optionally following a chronic liver injury, increasing hepatocyte function, or decreasing coagulation time in liver, in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a Wnt surrogate molecule, wherein the Wnt surrogate molecule is an agonist of a Wnt signaling pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. Schematic diagrams of illustrative formats of Wnt surrogate molecules.

FIGS. 2A-2D. Characterization of a Wnt surrogate molecule, R2M3-26.

FIGS. 3A-3D. Characterization of a Wnt surrogate molecule, R2M3-32.

FIGS. 4A-4B. Graphs showing that R2M3-26 and R2M3-32 activities can be inhibited by soluble Fzd ECD and by R2M3 IgG alone without the Lrp binding arm.

FIG. 5 . Characterization of illustrative R2M3-Lrp6 binder fusions in 293, Huh7, A375, and BNL.CL2 Wnt dependent reporter assays.

FIG. 6 . Characterization of illustrative 18R5-Lrp6 binder fusions in 293, A375, and BNL.CL2 Wnt dependent reporter assays.

FIG. 7 . Characterization of illustrative 18R5-Lrp5 binder fusions in 293 Wnt dependent reporter assays.

FIG. 8A-8B. Characterization of illustrative Fzd binders-Lrp6 binder 26 fusions in 293 Wnt dependent reporter assays.

FIG. 9 . SAR analysis of illustrative Wnt surrogate molecules in the IgG-Nab fusion format.

FIG. 10A-10B. Characterization of R2M3-26 in the Fab format in 293 Wnt dependent reporter assays.

FIG. 11A-11B. Characterization of R2M3-32 in the Fab format in 293 Wnt dependent reporter assays.

FIG. 12A-12B. Characterization of R2M3-26 in the Hetero-Ig format in 293 Wnt dependent reporter assays.

FIG. 13 . Characterization of 26-17SB9 in the VHH/sdAb-VHH/sdAb format, in different tandem formats, and on different ends of the Fc fragment in 293 Wnt dependent reporter assays.

FIG. 14A-14H. Characterization of 18R5-LRP6 Binder Fusions in tandem scFv formats in 293 Wnt dependent reporter assays.

FIG. 15A-15G. Characterization of various Wnt Surrogate molecules in the Fab-IgG format in 293 Wnt dependent reporter assays.

FIG. 16A-16C. Characterization of R2M3-26 in the F(ab′)2 format in 293 Wnt dependent reporter assays.

FIG. 17A-17H. Characterization of additional Wnt surrogate molecules in 293 Wnt dependent reporter assays

FIG. 18A-18C. A. Schematic diagram of the 2Fv-Ig format. B-C. Characterization of the Wnt surrogate molecule, 10SG11-1RC07.

FIG. 19 . Sequences of polypeptide chains of illustrative Wnt surrogates molecules.

FIG. 20A-20B. In vivo PK/PD characterization of R2M3-26

FIGS. 21A-21E. Images and graphs showing that systemic expression of 18R5-DKK1c for 14 days results in increased bone mineral density. *P value<0.05; ** P value<0.0001. For each time point, the bars from left to right are as follows: vehicle (diamond), romosozumab (square), AAV CAG-GFP (triangle), AAV ScFv (anti-GFP)-DKK1cF234K-Flag-His (inverted triangle), and AAV 18R5-DKK1c-FLagHis (circle).

FIGS. 22A-22D. Images and graphs showing that systemic expression of 18R5-DKK1c for 14 days or 28 days results in increased bone volume. For each time point, the bars from left to right are as follows: vehicle, romosozumab, AAV CAG-GFP, AAV ScFv (anti-GFP)-DKK1cF234K-Flag-His, and AAV 18R5-DKK1c-FLagHis. *P value<0.05; ** P value<0.0001, ****P value<0.0001.

FIGS. 23A-23B. Graphs showing the dynamic parameters of bone formation based on fluorochrome labelling. For each time point, the bars from left to right are as follows: vehicle, romosozumab, AAV CAG-GFP, AAV ScFv (anti-GFP)-DKK1cF234K-Flag-His, and AAV 18R5-DKK1c-FlagHis.

FIGS. 24A-24D. Graphs and images showing that systemic expression of 18R5-DKK1c results in increased osteoblast and reduced osteoclast on bone surface. For each time point, the bars from left to right are as follows: vehicle, romosozumab, AAV CAG-GFP, AAV ScFv (anti-GFP)-DKK1cF234K-Flag-His, and AAV 18R5-DKK1c-FlagHis. ** P value<0.05.

FIGS. 25A-25C. Diagram of assay for bone stiffness and fracture and graphs showing ultimate load to failure and stiffness in mice treated as indicated.

FIGS. 26A-26D. Graphs and images showing that systemic treatment with R2M3-26 results in rapid and sustained increase in bone after one week. For each timepoint, the bars from left to right correspond to the treatments indicated from top to bottom. **** indicates P value<0.0001.

FIGS. 27A-27C. Images and graphs showing that R2M3-26 treatment rapidly reverses the bone loss associated with ovariectomy-induced osteoporosis. For each time point, the bars from left to right correspond to the treatments indicated from top to bottom.

FIGS. 28A-28C. Images and graphs showing that a single injection of R2M3-26 rapidly increases bone volume.

FIGS. 29A-29D. Graphs showing that high doses of R2M3-26 and 1R-C07-26 significantly and rapidly increase bone formation in naïve mice. For each timepoint, the bars from left to right correspond to the treatments indicated from top to bottom.

FIG. 30 . Graphs showing that R2M3-26 and 1R-C07-3 increase bone mineral density in naïve mice. For each timepoint, the bars from left to right correspond to the treatments indicated from top to bottom.

FIG. 31 . Graph showing changes in whole body bone mineral density (BMD) measured weekly in ovariectomized mice as compared with naïve and sham surgery operated mice.

FIG. 32 . Changes in vertebral mineral density (image shown) and changes the vertebral resistance to compression fracture in vertebra isolated from mice after various treatments (bar graph) as measured in newton units of force (N) after 4 weeks of treatment.

FIGS. 33A-D. Test of Wnt surrogate molecules in an Einhorn fracture model. Radiographs of the callus after 1 week (A) and 6 weeks (B) of treatment with Wnt surrogate molecules are shown. Graphs of changes in whole body bone mineral density (BMD) in contralateral femur are shown (C). Scatter plots showing changes in callus tissue volume, callus bone volume, bone volume/tissue volume ratio (BV/TV), and bone mineral content per millimeter (BMC/mm) as shown in (D) along with representative images of bone slices are shown.

FIG. 34 . Graph with changes in whole body bone mineral density (BMD) measured weekly with different Wnt surrogate molecule dosing schedules is shown.

FIG. 35 . Graph with changes in whole body bone mineral density (BMD) measured weekly from mice treated with different Wnt surrogate molecule alone and in combination with Romosozumab is shown.

FIG. 36 . Levels of therapeutic molecules in serum as measured by ELISA. These data accompany gene expression data presented in Table 4.

FIGS. 37A-37C. Liver (A), small intestine (B) and colon (C) to body weight ratio after treatment with AAV-delivered Wnt surrogates. (**) p<0.01. For each graph, the treatments shown from left to right correspond to those in the legend from top to bottom.

FIGS. 38A-38B. Body weight (A) and liver to body weight (B) ratio after treatment with recombinantly produced Wnt surrogate proteins. (*) p<0.05. For each time point, the treatments shown from left to right correspond to those in the legend from top to bottom.

FIGS. 39A-39D. Induction of proliferation markers in response to R2M3-26 and Rspo2 recombinant proteins. Liver Ki67 (A) and cyclinD1 (B) mRNA expression. Average count of PCNA (C) or phospho-histone H3 (D) positive nuclei per 10× field after immunohistochemistry staining with PCNA and phospho-histone H3 antibodies respectively. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. For each time point, the treatments shown from left to right correspond to those in the legend from top to bottom.

FIGS. 40A-40H. Efficacy of AAV-delivered Wnt surrogate and R-Spondin in a thioacetamide-induced chronic liver disease model. Design for study 1 (A) and study 2 (B). Liver to body weight ratio (C-D), liver weight (E-F), liver collagen A1 mRNA expression (G) and percentage red area in liver histological sections stained with Sirius red (H) in response to AAV-delivered wnt surrogate and R-spondin in study 1 (C, E, G and H) and study 2 (D, F, H). (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. For each graph, the treatments shown from left to right correspond to those in the legend from top to bottom (not including baseline).

FIGS. 41A-41N. Efficacy of recombinantly produced Wnt surrogate and R-Spondin in a thioacetamide-induced chronic liver disease model. Study design (A). D-2, D0, D3, D7, D10, D14 represents days relative to the start of treatment with recombinant proteins. Liver to body weight ratio (B, C), liver axin2 mRNA (D, E), cyclinD1 mRNA (F, G) and Ki67 mRNA (H, I) expression, average count of PCNA (J, K) or phospho-histone H3 (L, M) positive nuclei per 10× field after immunohistochemistry staining with PCNA and phospho-histone H3 antibodies respectively, in a study using Rspo2 mono treatment (B, D, F, H, J, L) or R2M3-26/Rspo2 combination treatment (C, E, G, I, K, M). Pro-thrombin time ratio relative to the average pro-thrombin time in plasma collected from control naïve mice without TAA exposure (N). (*) p<0.05, (**) p<0.01, (****) p<0.0001. No TAA treatment is indicated by the dashed line. For each bar graph timepoint, the treatments shown from left to right correspond to those in the legend shown above from top to bottom.

FIGS. 42A-42C. Efficacy of recombinantly produced Wnt surrogate and R-Spondin in a CC14-induced chronic liver disease model. Study design (A). Liver to body weight ratio (B) pro-thrombin time (C) and sirius red staining (D) in response to CC14 treatment, R2M3-26 and Rspo2. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001. For each graph, the treatments shown from left to right correspond to those in the legend from top to bottom (not including baseline).

FIGS. 43A-43D. Induction of proliferation markers in response to recombinantly produced Wnt surrogate in an acetaminophen-induced acute liver injury model. Study design (A). Serum level of alanine transferase at 24 and 48 hours after treatment with acetaminophen (B). Relative cyclinD1 (C) and Ki67 (D) mRNA expression. (*) p<0.05, (***) p<0.001, (****) p<0.0001.

FIGS. 44A-44D. Induction of proliferation markers in response to R-Spondin in an acetaminophen-induced acute liver injury model. Study design (A). Serum level of alanine transferase at 24 and 48 hours after treatment with acetaminophen (B). Relative cyclinD1 (C) and Ki67 (D) mRNA expression. (**) p<0.01, (***) p<0.001, (****) p<0.0001.

FIGS. 45A-45D. Induction of proliferation markers in response to Wnt surrogate and R-Spondin in an acetaminophen-induced acute liver injury model. Study design (A). Serum level of alanine transferase at 24, 36, 48 and 60 hours after treatment with acetaminophen (B). Relative cyclinD1 (C) and Ki67 (D) mRNA expression. (*) p<0.05, (****) p<0.0001. For each time point, the treatments shown from left to right correspond to those in the legend from top to bottom.

FIGS. 46A-46D. Efficacy of recombinantly produced Wnt surrogate and R-Spondin on the survival of mice after acetaminophen-induced liver injury. Study design (A). Survival curve of mice treated with the control anti-eGFP control protein or R2M3-26 (B), Rspo2 (C) or a combination of R2M3-26 and Rspo2 (D) recombinant proteins.

DETAILED DESCRIPTION

The present disclosure relates to Wnt surrogate molecules that bind to one or more Fzd receptor and one or more LRP5 or LRP6 receptor and modulate a downstream Wnt signaling pathway. In particular embodiments, the Wnt surrogate molecules activate a Wnt signaling pathway or increase signaling via a Wnt signaling pathway. In particular embodiments, the Wnt surrogate molecules disclosed herein comprise: (i) one or more antibodies or antigen-binding fragments thereof that specifically bind to one or more Fzd receptor, including antibodies or antigen-binding fragments thereof having particular Fzd receptor specificity and/or functional properties; and (ii) one or more antibodies or antigen-binding fragments thereof that specifically bind to LRP5 and/or LRP6. Certain embodiments encompass specific structural formats or arrangements of the Fzd binding region(s) and LRP5/6 binding region(s) of Wnt surrogate molecules advantageous in increasing downstream Wnt pathway signaling and related biological effects.

Embodiments of the invention pertain to the use of Wnt surrogate molecules for the diagnosis, assessment and treatment of diseases and disorders associated with Wnt signaling pathways. In certain embodiments, the subject Wnt surrogate molecules are used to modulate a Wnt signaling pathway in a cell or tissue. In certain embodiments, the subject Wnt surrogate molecules are used in the treatment or prevention of diseases and disorders associated with aberrant or deregulated (e.g., reduced) Wnt signaling, or for which modulating, e.g., increasing, Wnt signaling would provide a therapeutic benefit.

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

Embodiments of the present invention relate to antibodies and antigen-binding fragments thereof that bind to one or more Fzd receptor. Sequences of illustrative antibodies, or antigen-binding fragments, or complementarity determining regions (CDRs) thereof, are set forth in SEQ ID NOs:1-65 or 129-132, Tables 1A and 1B, and Table 3. Anti-Fzd antibodies and antigen-binding fragments there that may be used or present in the Wnt surrogate molecules disclosed herein include, but are not limited to, those described in the U.S. provisional application No. 62/607,877, titled Anti-Frizzled Antibodies and Methods of Use, filed on Dec. 19, 2017.

Embodiments of the present invention relate to antibodies and antigen-binding fragments thereof that bind to LRP5 and/or LRP6. Sequences of illustrative antibodies, or antigen-binding fragments, or complementarity determining regions (CDRs) thereof, are set forth in SEQ ID NOs:66-88 or 133, Tables 2A and 2B, and Table 3. Anti-LRP5/6 antibodies and antigen-binding fragments there that may be used or present in the Wnt surrogate molecules disclosed herein include, but are not limited to, those described in the U.S. provisional application No. 62/607,879, titled Anti-LRP5/6 Antibodies and Methods of Use, filed on Dec. 19, 2017.

As is well known in the art, an antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab′, F(ab′)2, Fv), single chain (scFv), VHH or sdAb, synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody or an antigen-binding fragment thereof, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity. “Diabodies” or 2scFV-Ig antibodies, are multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also a particular form of antibody contemplated herein. Minibodies comprising a scFv joined to a CH3 domain are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). See e.g., Ward, E. S. et al., Nature 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); PCT/US92/09965; WO94/13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996.

The term “antigen-binding fragment” as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chain, or of a VHH or sdAb, that binds to the antigen of interest, in particular to one or more Fzd receptor or LRP5 or LRP6 receptor. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set forth herein from antibodies that bind one or more Fzd receptor or LRP5 and/or LRP6. In particular embodiments, an antigen-binding fragment may comprise all three VH CDRs or all three VL CDRs. Similarly, an antigen binding fragment thereof may comprise all three CDRs of a VHH or sdAb. An antigen-binding fragment of a Fzd-specific antibody is capable of binding to a Fzd receptor. An antigen-binding fragment of a LRP5/6-specific antibody is capable of binding to a LRP5 and/or LRP6 receptor. As used herein, the term encompasses not only isolated fragments but also polypeptides comprising an antigen-binding fragment of an antibody disclosed herein, such as, for example, fusion proteins comprising an antigen-binding fragment of an antibody disclosed herein, such as, e.g., a fusion protein comprising a VHH or sdAb that binds one or more Fzd receptors and a VHH or sdAb that binds LRP5 and/or LRP6.

The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a Wnt surrogate molecule or binding region thereof) is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In certain embodiments, a Wnt surrogate molecule or binding region thereof (e.g., an antibody or antigen-binding fragment thereof) is said to specifically bind an antigen when the equilibrium dissociation constant is ≤10⁻⁷ or ≤10⁻⁸ M. In some embodiments, the equilibrium dissociation constant may be ≤10⁻⁹ M or ≤10⁻¹⁰ M.

In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term “CDR set” refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain “canonical” structures—regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

The structures and locations of immunoglobulin CDRs and variable domains may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (immuno.bme.nwu.edu). Alternatively, CDRs may be determined by using “IMGT®, the international ImMunoGeneTics information System® available at www.imgt.org (see, e.g., Lefranc, M.-P. et al. (1999) Nucleic Acids Res., 27:209-212; Ruiz, M. et al. (2000) Nucleic Acids Res., 28:219-221; Lefranc, M.-P. (2001) Nucleic Acids Res., 29:207-209; Lefranc, M.-P. (2003) Nucleic Acids Res., 31:307-310; Lefranc, M.-P. et al. (2004) In Silico Biol., 5, 0006 [Epub], 5:45-60 (2005)]; Lefranc, M.-P. et al. (2005) Nucleic Acids Res., 33:D593-597; Lefranc, M.-P. et al. (2009) Nucleic Acids Res., 37:D1006-1012; Lefranc, M.-P. et al. (2015) Nucleic Acids Res., 43:D413-422).

A “monoclonal antibody” refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term “monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv), VHH or sdAb, variants thereof, fusion proteins comprising an antigen-binding fragment of a monoclonal antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope, including Wnt surrogate molecules disclosed herein. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody”.

The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab′)2 fragment which comprises both antigen-binding sites. An Fv fragment for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V_(H)::V_(L) heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (III et al., Prot. Eng. 10: 949-57 (1997); minibodies (Martin et al., EMBO J 13: 5305-9 (1994); diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins (Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al., Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity. In still other embodiments, bispecific or chimeric antibodies may be made that encompass the ligands of the present disclosure. For example, a chimeric antibody may comprise CDRs and framework regions from different antibodies, while bispecific antibodies may be generated that bind specifically to one or more Fzd receptor through one binding domain and to a second molecule through a second binding domain. These antibodies may be produced through recombinant molecular biological techniques or may be physically conjugated together.

A single chain Fv (scFv) polypeptide is a covalently linked V_(H)::V_(L) heterodimer which is expressed from a gene fusion including V_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated—but chemically separated—light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

In certain embodiments, an antibody as described herein is in the form of a diabody. Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).

A dAb fragment of an antibody consists of a V_(H) domain (Ward, E. S. et al., Nature 341, 544-546 (1989)).

Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This proprietary antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one area on the UniBody® that can bind to cognate antigens (e.g., disease targets) and the UniBody® therefore binds univalently to only one site on target cells.

In certain embodiments, the antibodies of the present disclosure may take the form of a VHH or sdAb. VHH or sdAb technology was originally developed following the discovery and identification that camelidae (e.g., camels and llamas) possess fully functional antibodies that consist of heavy chains only and therefore lack light chains. These heavy-chain only antibodies contain a single variable domain (V_(HH)) and two constant domains (C_(H)2, C_(H)3). The cloned and isolated single variable domains have full antigen binding capacity and are very stable. These single variable domains, with their unique structural and functional properties, form the basis of “VHH or sdAb”. VHH or sdAb are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g. U.S. Pat. No. 6,765,087), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces, Kluyvermyces, Hansenula or Pichia (see e.g. U.S. Pat. No. 6,838,254). The production process is scalable and multi-kilogram quantities of VHH or sdAb have been produced. VHH or sdAb may be formulated as a ready-to-use solution having a long shelf life. The VHH or sdAb method (see, e.g., WO 06/079372) is a proprietary method for generating VHH or sdAb against a desired target, based on automated high-throughput selection of B-cells. VHH or sdAb are single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies. VHH antibodies or sdAb, typically have a small size of around 15 kDa.

In certain embodiments, the antibodies or antigen-binding fragments thereof as disclosed herein are humanized. This refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains. Epitope binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A. F. et al., (1989) Proc Natl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). Illustrative methods for humanization of the anti-Fzd antibodies disclosed herein include the methods described in U.S. Pat. No. 7,462,697.

Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular epitope, the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K., et al., (1993) Cancer Res 53:851-856. Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al., (1988) Science 239:1534-1536; Kettleborough, C. A., et al., (1991) Protein Engineering 4:773-3783; Maeda, H., et al., (1991) Human Antibodies Hybridoma 2:124-134; Gorman, S. D., et al., (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, P. R., et al., (1991) Bio/Technology 9:266-271; Co, M. S., et al., (1991) Proc Natl Acad Sci USA 88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA 89:4285-4289; and Co, M. S. et al., (1992) J Immunol 148:1149-1154. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies of the present disclosure may be chimeric antibodies. In this regard, a chimeric antibody is comprised of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes. As noted above with regard to humanized antibodies, the antigen-binding fragment of a chimeric antibody may comprise only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may comprise an entire variable domain (VL, VH or both).

Wnt Surrogates

The disclosure provides, in certain aspects, Wnt surrogate molecules that bind both one or more Fzd receptors and one or both of LRP5 and/or LRP6. Wnt surrogate molecules may also be referred to as “Wnt surrogates” or “Wnt mimetics.” In particular embodiments, the Wnt surrogate molecules bind one or more human Fzd receptors and one or both of a human LRP5 and/or a human LRP6.

In certain embodiments, a Wnt surrogate molecule is capable of modulating or modulates Wnt signaling events in a cell contacted with the Wnt surrogate molecule. In certain embodiments, the Wnt surrogate molecule increases Wnt signaling, e.g., via the canonical Wnt/β-catenin pathway. In certain embodiments, the Wnt surrogate molecule specifically modulates the biological activity of a human Wnt signaling pathway.

Wnt surrogate molecules of the present invention are biologically active in binding to one or more Fzd receptor and to one or more of LRP5 and LRP6, and in activation of Wnt signaling, i.e., the Wnt surrogate molecule is a Wnt agonist. The term “Wnt agonist activity” refers to the ability of an agonist to mimic the effect or activity of a Wnt protein binding to a frizzled protein and/or LRP5 or LRP6. The ability of the Wnt surrogate molecules and other Wnt agonists disclosed herein to mimic the activity of Wnt can be confirmed by a number of assays. Wnt agonists typically initiate a reaction or activity that is similar to or the same as that initiated by the receptor's natural ligand. In particular, the Wnt agonists disclosed herein activate, enhance or increase the canonical Wnt/β-catenin signaling pathway. As used herein, the term “enhances” refers to a measurable increase in the level of Wnt/β-catenin signaling compared with the level in the absence of a Wnt agonist, e.g., a Wnt surrogate molecule disclosed herein. In particular embodiments, the increase in the level of Wnt/β-catenin signaling is at least 10%, at least 20%, at least 50%, at least two-fold, at least five-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold as compared to the level of Wnt/β-catenin signaling in the absence of the Wnt agonist, e.g., in the same cell type. Methods of measuring Wnt/β-catenin signaling are known in the art and include those described herein.

In particular embodiments, Wnt surrogate molecules disclosed herein are bispecific, i.e., they specifically bind to two or more different epitopes, e.g., one or more Fzd receptor, and LRP5 and/or LRP6.

In particular embodiments, Wnt surrogate molecules disclosed herein are multivalent, e.g., they comprise two or more regions that each specifically bind to the same epitope, e.g., two or more regions that bind to an epitope within one or more Fzd receptor and/or two or more regions that bind to an epitope within LRP5 and/or LRP6. In particular embodiments, they comprise two or more regions that bind to an epitope within one or more Fzd receptor and two or more regions that bind to an epitope within LRP5 and/or LRP6. In certain embodiments, Wnt surrogate molecules comprise a ratio of the number of regions that bind one or more Fzd receptor to the number of regions that bind LRP5 and/or LRP6 of or about: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 2:3, 2:5, 2:7, 7:2, 5:2, 3:2, 3:4, 3:5, 3:7, 3:8, 8:3, 7:3, 5:3, 4:3, 4:5, 4:7, 4:9, 9:4, 7:4, 5:4, 6:7, 7:6, 1:2, 1:3, 1:4, 1:5, or 1:6. In certain embodiments, Wnt surrogate molecules are bispecific and multivalent.

Wnt surrogate molecules disclosed herein may have any of a variety of different structural formats or configurations. Wnt surrogate molecules may comprise polypeptides and/or non-polypeptide binding moieties, e.g., small molecules. In particular embodiments, Wnt surrogate molecules comprise both a polypeptide region and a non-polypeptide binding moiety. In certain embodiments, Wnt surrogate molecules may comprise a single polypeptide, or they may comprise two or more, three or more, or four or more polypeptides. In certain embodiments, one or more polypeptides of a Wnt surrogate molecule are antibodies or antigen-binding fragments thereof. In certain embodiments, Wnt surrogates comprise two antibodies or antigen binding fragments thereof, one that binds one or more Fzd and one that binds LRP5 and/or LRP6. In certain embodiments, the Wnt surrogates comprises one, two, three, or four polypeptides, e.g., linked or bound to each other or fused to each other.

When the Wnt surrogate molecules comprise a single polypeptide, they may be a fusion protein comprising one or more Fzd binding domain and one or more LRP5/6 binding domain. The binding domains may be directly fused or they may be connected via a linker, e.g., a polypeptide linker, including but not limited to any of those disclosed herein.

When the Wnt surrogate molecules comprise two or more polypeptides, the polypeptides may be linked via covalent bonds, such as, e.g., disulfide bonds, and/or noncovalent interactions. For example, heavy chains of human immunoglobulin IgG interact at the level of their CH3 domains directly, whereas, at the level of their CH2 domains, they interact via the carbohydrate attached to the asparagine (Asn) N84.4 in the DE turn. In particular embodiments, the Wnt surrogate molecules comprise one or more regions derived from an antibody or antigen-binding fragment thereof, e.g., antibody heavy chains or antibody light chains or fragments thereof. In certain embodiments, a Wnt surrogate polypeptide comprises two antibody heavy chain regions (e.g., hinge regions) bound together via one or more disulfide bond. In certain embodiments, a Wnt surrogate polypeptide comprises an antibody light chain region (e.g., a CL region) and an antibody heavy chain region (e.g., a C_(H)1 region) bound together via one or more disulfide bond.

Wnt surrogate polypeptides may be engineered to facilitate binding between two polypeptides. For example, Knob-into-holes amino acid modifications may be introduced into two different polypeptides to facilitate their binding. Knobs-into-holes amino acid (AA) changes is a rational design strategy developed in antibody engineering, used for heterodimerization of the heavy chains, in the production of bispecific IgG antibodies. AA changes are engineered in order to create a knob on the C_(H)3 of the heavy chains from a first antibody and a hole on the C_(H)3 of the heavy chains of a second antibody. The knob may be represented by a tyrosine (Y) that belongs to the ‘very large’ IMGT volume class of AA, whereas the hole may be represented by a threonine (T) that belongs to the ‘small’ IMGT volume class. Other means of introducing modifications into polypeptides to facilitate their binding are known and available in the art. For example, specific amino acids may be introduced and used for cross-linking, such as Cysteine to form an intermolecular disulfide bond.

Wnt surrogate molecules may have a variety of different structural formats, including but not limited to those shown in FIG. 1 .

In one embodiment, a Wnt surrogate molecule comprises an scFv or antigen-binding fragment thereof fused to a VHH or sdAb or antigen-binding fragment thereof. In certain embodiments, the scFv specifically binds one or more Fzd receptor, and the VHH or sdAb specifically binds LRP5 and/or LRP6. In certain embodiments, the scFv specifically binds LRP5 and/or LRP6, and the VHH or sdAb specifically binds one or more Fzd receptor. In particular embodiments, the scFv or antigen-binding fragment thereof is fused directly to the VHH or sdAb or antigen-binding fragment thereof, whereas in other embodiments, the two binding regions are fused via a linker moiety. In particular embodiments, the VHH or sdAb is fused to the N-terminus of the scFV, while in other embodiments, the VHH or sdAb is fused to the C-terminus of the scFv. In particular embodiments, the scFv is described herein or comprises any of the CDR sets described herein. In particular embodiments, the VHH or sdAb is described herein or comprises any of the CDR sets disclosed herein.

In various embodiments, including but not limited to those depicted in FIG. 1A, a Wnt surrogate molecule comprises one or more Fab or antigen-binding fragment thereof and one or more VHH or sdAb or antigen-binding fragment thereof (or alternatively, one or more scFv or antigen-binding fragment thereof). In certain embodiments, the Fab specifically binds one or more Fzd receptor, and the VHH or sdAb (or scFv) specifically binds LRP5 and/or LRP6. In certain embodiments, the Fab specifically binds LRP5 and/or LRP6, and the VHH or sdAb (or scFv) specifically binds one or more Fzd receptor. In certain embodiments, the VHH or sdAb (or scFv) is fused to the N-terminus of the Fab, while in some embodiments, the VHH or sdAb (or scFv) is fused to the C-terminus of the Fab. In particular embodiments, the Fab is present in a full IgG format, and the VHH or sdAb (or scFv) is fused to the N-terminus and/or C-terminus of the IgG light chain. In particular embodiments, the Fab is present in a full IgG format, and the VHH or sdAb (or scFv) is fused to the N-terminus and/or C-terminus of the IgG heavy chain. In particular embodiments, two or more VHH or sdAb (or scFvs) are fused to the IgG at any combination of these locations.

Fabs may be converted into a full IgG format that includes both the Fab and Fc fragments, for example, using genetic engineering to generate a fusion polypeptide comprising the Fab fused to an Fc region, i.e., the Fab is present in a full IgG format. The Fc region for the full IgG format may be derived from any of a variety of different Fcs, including but not limited to, a wild-type or modified IgG1, IgG2, IgG3, IgG4 or other isotype, e.g., wild-type or modified human IgG1, human IgG2, human IgG3, human IgG4, human IgG4Pro (comprising a mutation in core hinge region that prevents the formation of IgG4 half molecules), human IgA, human IgE, human IgM, or the modified IgG1 referred to as IgG1 LALAPG. The L₂₃₅A, P₃₂₉G (LALA-PG) variant has been shown to eliminate complement binding and fixation as well as Fc-γ dependent antibody-dependent cell-mediated cytotoxity (ADCC) in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an “effectorless” antibody framework scaffold between mice and primates. In particular embodiments of any of the IgG disclosed herein, the IgG comprises one or more of the following amino acid substitutions: N297G, N297A, N297E, L234A, L235A, or P236G.

Non-limiting examples of bivalent and bispecific Wnt surrogate molecules that are bivalent towards both the one or more Fzd receptor and the LRP5 and/or LRP6 are provided as the top four structures depicted in FIG. 1A, where the VHH or sdAb or scFv is depicted in white, and the Fab or IgG is depicted in black. As shown, the VHH or sdAb (or scFvs) may be fused to the N-termini of both light chains, to the N-termini of both heavy chains, to the C-termini of both light chains, or to the C-termini of both heavy chains. It is further contemplated, e.g., that VHH or sdAb (or scFvs) could be fused to both the N-termini and C-termini of the heavy and/or light chains, to the N-termini of the light chains and the heavy chains, to the C-termini of the heavy and light chains, to the N-termini of the heavy chains and C-termini of the light chains, or to the C-termini of the heavy chains and the N-termini of the light chains. In other related embodiments, two or more VHH or sdAb (or scFvs) may be fused together, optionally via a linker moiety, and fused to the Fab or IgG at one or more of these locations. In a related embodiment, the Wnt surrogate molecule has a Hetero-IgG format, whereas the Fab is present as a half antibody, and one or more VHH or sdAb (or scFv) is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, the C-terminus of the Fc, or the C-terminus of the Fab. A bispecific but monovalent to each receptor version of this format is depicted at the bottom of FIG. 1A. In certain embodiments, the Fab or antigen-binding fragment (or IgG) thereof is fused directly to the VHH or sdAb (or scFv) or antigen-binding fragment thereof, whereas in other embodiments, the binding regions are fused via a linker moiety. In particular embodiments, the Fab is described herein or comprises any of the CDR sets described herein. In particular embodiments, the VHH or sdAb or scFv is described herein or comprises any of the CDR sets disclosed herein.

In various embodiments, including but not limited to those depicted in FIG. 1B, a Wnt surrogate molecule comprises one or more Fab or antigen-binding fragment thereof that binds one or more Fzd receptor and one or more Fab or antigen-binding fragment thereof that binds LRP5 and/or LRP6. In certain embodiments, it comprises two Fab or antigen-binding fragments thereof that bind one or more Fzd receptor and/or two Fab or antigen-binding fragments thereof that bind LRP5 and/or LRP6. In particular embodiments, one or more of the Fab is present in a full IgG format, and in certain embodiments, both Fab are present in a full IgG format. In certain embodiments, the Fab in full IgG format specifically binds one or more Fzd receptor, and the other Fab specifically binds LRP5 and/or LRP6. In certain embodiments, the Fab specifically binds one or more Fzd receptor, and the Fab in full IgG format specifically binds LRP5 and/or LRP6. In certain embodiments, the Fab specifically binds LRP5 and/or LRP6, and the Fab in full IgG format specifically binds one or more Fzd receptor. In certain embodiments, the Fab is fused to the N-terminus of the IgG, e.g., to the heavy chain or light chain N-terminus, optionally via a linker. In certain embodiments, the Fab is fused to the N-terminus of the heavy chain of the IgG and not fused to the light chain. In particular embodiments, the two heavy chains can be fused together directly or via a linker. An example of such a bispecific and bivalent with respect to both receptors is shown at the top of FIG. 1B. In other related embodiments, two or more VHH or sdAb may be fused together, optionally via a linker moiety, and fused to the Fab or IgG at one or more of these locations. In a related embodiment, the Wnt surrogate molecule has a Hetero-IgG format, whereas one of the Fab is present as a half antibody, and the other Fab is fused to one or more of the N-terminus of the Fc, the N-terminus of the Fab, or the C-terminus of the Fc. A bispecific but monovalent to each receptor version of this format is depicted at the bottom of FIG. 1B. In certain embodiments, the Fab or antigen-binding fragment thereof is fused directly to the other Fab or IgG or antigen-binding fragment thereof, whereas in other embodiments, the binding regions are fused via a linker moiety. In particular embodiments, the one or both of the two Fabs are described herein or comprise any of the CDR sets described herein.

In certain embodiments, Wnt surrogate molecules have a format as described in PCT Application Publication No. WO2017/136820, e.g., a Fabs-in-tandem IgG (FIT-IG) format. Shiyong Gong, Fang Ren, Danqing Wu, Xuan Wu & Chengbin Wu (2017). FIT-IG also include the formats disclosed in “Fabs-in-tandem immunoglobulin is a novel and versatile bispecific design for engaging multiple therapeutic targets” mAbs, 9:7, 1118-1128, DOI: 10.1080/19420862.2017.1345401. In certain embodiments, FIT-IGs combine the functions of two antibodies into one molecule by re-arranging the DNA sequences of two parental monoclonal antibodies into two or three constructs and co-expressing them in mammalian cells. Examples of FIT-IG formats and constructs are provided in FIGS. 1A and 1B and FIGS. 2A and 2B of PCT Application Publication No. WO2017/136820. In certain embodiments, FIT-IGs require no Fc mutation; no scFv elements; and no linker or peptide connector. The Fab-domains in each arm work “in tandem” forming a tetravalent bi-specific antibody with four active and independent antigen binding sites that retain the biological function of their parental antibodies In particular embodiments, Wnt surrogates comprises a Fab and an IgG. In certain embodiments, the Fab binder LC is fused to the HC of the IgG, e.g., by a linker of various length in between. In various embodiment, the Fab binder HC can be fused or unfused to the LC of the IgG. A variation of this format has been called Fabs-in-tandem IgG (or FIT-Ig).

In particular embodiments, Wnt surrogate molecules comprise two or more VHH or sdAb (or scFvs), including at least one that binds one or more Fzd receptor and at least one that binds LRP5 and/or LRP6. In certain embodiments, one of the binding regions is a VHH or sdAb and the other is an scFv. Wnt memetic molecules comprising two or more VHH or sdAb (or scFvs) may be formatted in a variety of configurations, including but not limited to those depicted in FIG. 1C. In certain bispecific, bivalent formats, two or more VHH or sdAb (or scFvs) are fused in tandem or fused to two different ends of an Fc, optionally via one or more linkers. Where linkers are present, the linker and its length may be the same or different between the VHH or sdAb (or scFv) and the other VHH or sdAb (or scFv), or between the VHH or sdAb and Fc. For example, in certain embodiments, the VHH or sdAb is fused to the N-terminus and/or C-terminus of the IgG heavy chain. In particular embodiments, two or more VHH or sdAb are fused to the IgG at any combination of these locations. Non-limiting examples of bivalent and bispecific Wnt surrogate molecules of this format are depicted as the top seven structures depicted in FIG. 1C, where the first VHH or sdAb is depicted in white, the Fc or IgG is depicted in black, and the second VHH or sdAb is depicted as light gray. In various embodiments, both VHH or sdAb may be fused to the N-termini of the Fc, to the C-termini of the Fc, or one or more VHH or sdAb may be fused to either or both of an N-terminus or C-terminus of the Fc. In a related embodiment, the Wnt surrogate molecule has a Hetero-IgG format, whereas one VHH or sdAb is present as a half antibody, and the other is fused to the N-terminus of the Fc or the C-terminus of the Fc. A bispecific but monovalent to each receptor version of this format is depicted at the bottom of FIG. 1C. In certain embodiments, the VHH or sdAb is fused directly to the other VHH or sdAb whereas in other embodiments, the binding regions are fused via a linker moiety. In particular embodiments, the VHH or sdAb are described herein or comprises any of the CDR sets described herein. In various embodiments, any of these formats may comprise one or more scFvs in place of one or more VHH or sdAb.

In certain embodiments, a Wnt surrogate molecule is formatted as a diabody. As shown in FIG. 1D, the binders against Fzd and LRP can also be linked together in a diabody (or DART) configuration. The diabody can also be in a single chain configuration. If the diabody is fused to an Fc, this will create a bivalent bispecific format. Without fusion to Fc, this would be a monovalent bispecific format. In certain embodiments; a diabody is a noncovalent dimer scFv fragment that consists of the heavy-chain variable (VH) and light-chain variable (VL) regions connected by a small peptide linker. Another form of diabody is a single-chain (Fv)2 in which two scFv fragments are covalently linked to each other.

As discussed, Wnt surrogate molecules, in various embodiments, comprise one or more antibodies or antigen-binding fragments thereof disclosed herein. Thus, in particular embodiments, a Wnt surrogate comprises two polypeptides, wherein each polypeptide comprises an Nab or scFv that binds LRP5/6 and an Nab or scFv that binds one or more Wnts, optionally wherein one of the binding domains is an scFv and the other is an Nab. In certain embodiments, a Wnt surrogate comprises three polypeptides, wherein the first polypeptide comprises an antibody heavy chain and the second polypeptide comprises an antibody light chain, wherein the antibody heavy chain and light chain bind LRP5/6 or one or more Fzds, and wherein the third polypeptide comprises a VHH or sdAb fused to a heavy chain Fc region, wherein the VHH or sdAb binds to either LRP5/6 or one or more Fzds. In other embodiments, Wnt polypeptides comprise four polypeptides, including two heavy chain polypeptides and two light chain polypeptides, wherein the two heavy chains and two light chains bind LRP5/6 or one or more Fzds, and further comprise one or more Nab or scFv fused to one or more of the heavy chains and/or light chains, wherein the Nab or scFv binds to LRP5/6 or one or more Fzds. In another illustrative embodiment, a Wnt surrogate comprises at least four polypeptides, including two heavy chain polypeptides and two light chain polypeptides that bind either LRP5/6 or one or more Fzds, wherein the Wnt surrogate further comprises a Fab that binds either LRP5/6 or one or more Fzds. For example, the Fab may comprise two polypeptides, each fused to one of the two heavy chain polypeptides, and two polypeptides, each fused to one of the two light chain polypeptides, or it may comprise two polypeptides each fused to one of the two heavy chain polypeptides and two additional polypeptides, each bound to one of the two polypeptides fused to the heavy chain polypeptides, thus making a second Fab. Other configurations may be used to produce the Wnt surrogates disclosed herein.

In particular embodiments, a Wnt surrogate molecule comprises a Fzd binding region, e.g., an anti-Fzd antibody, or antigen-binding fragment thereof, fused or bound to a polypeptide that specifically binds to one or more Fzd receptor. In particular embodiments, the polypeptide that specifically binds to one or more Fzd receptor is an antibody or antigen-binding fragment thereof. If certain embodiments, it is an antibody or antigen-binding fragment thereof disclosed herein or in the U.S. provisional patent application Ser. No. 62/607,877, titled, “Anti-Frizzled antibodies and Methods of Use,” filed on Dec. 19, 2017, which is incorporated herein by reference in its entirety. In particular embodiments, the Fzd binding domain comprises the three heavy chain CDRs and/or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more Fzd receptor provided in Table 1A. In particular embodiments, the Fzd binding domain comprises the three heavy chain CDRs and/or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more Fzd receptor provided in Table 1A, wherein the CDRs collectively comprise one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., substitutions, deletions, or additions. In certain embodiments, the Fzd binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so Table 1A only includes the three heavy chain CDRs. In particular embodiments, the Fzd binding domain comprises the three CDR HC sequences provided in Table 1A or variants wherein the CDRs collectively comprise one, two, three, four, five, six, seven or eight amino acid modifications. In particular embodiments, the Fzd binding domain comprises the heavy chain fragment and/or light chain fragment of any of the illustrative antibodies or fragments thereof that bind to one or more Fzd receptor provided in Table 1B or SEQ ID NOs:1-65 or 129-132 (or an antigen-binding fragment or variant of either). In certain embodiments, the Fzd binding domain is an Fab or was derived from an Fab, so the heavy chain of Table 1B includes VH and CH1 sequence, but not CH2 or CH3 sequences. In certain embodiments, the Fzd binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so Table 1B includes the VHH domain. In certain embodiments, the Fzd binding region is a polypeptide, e.g., an antibody or antigen-binding fragment thereof, that competes with any of these antibodies for binding to one or more Fzd receptor.

TABLE 1A Anti-Fzd Antibody Clone IDs and CDR sequences CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 Clone Initial SEQ SEQ SEQ SEQ SEQ SEQ ID Binding CDRH1 ID CDRH2 ID CDRH3 ID CDRL1 ID CDRL2 ID CDRL3 ID 00 Fzd YTFTS 391 GWIS 570 CARASAW 752 SGSSSNI 1156 SNY 1256 CAAW 1265 1S 1 YGIS AYNG TPYGAFDI GSHTVS QRP DGSLF - NTNY W S GHWV A0 A T 1 00 Fzd GSISS 283 GSIYH 547 CARFYYDIL 818 RSSRSLL 1142 TLS 1259 CMQSI 1295 1S 1 GGYS SGSTY TGYSYFDY DTDDGN HRA QLPW - WS YN W TYLD S TF B0 1 00 Fzd GSISN 282 GEIDR 488 CARVRARR 945 SGNTLGS 1155 QDS 1246 CQVW 1431 1S 1 YYWS SGDT FLVSDRSAF HYVS KRP DSSTV - NYN DIW S VF E0 1 00 Fzd GSISG 281 GSIYF 546 CARVMLIT 942 RSSQSLL 1138 LGS 1237 CMQG 1289 1S 1 NNYY TGGT DAFDIW HSNGYN NRA THWP - ZG YYN YLD S YTF F0 1 00 Fzd GSISS 285 GYIYY 589 CARATYGG 760 TRSSSNI 1161 GNS 1220 CGTW 1267 1S 1 SSYY SGSTY GAGYDV IRPS DSSLS - WG YN DAFDIW AWVF G H 01 00 Fzd GSISS 284 GYIYY 589 CARHAGFY 875 RSSQSLL 1138 LGS 1236 CMQA 1280 1S 1 GGYY SGSTY GLADYFDY HSNGYN KRA LQIPP - WS YN W YLD S TF H0 1 00 Fzd GSISS 284 GYIYY 589 CARGKGYS 845 QASQDI 1041 DAS 1185 CQQN 1332 1S 1 GGYY SGSTY YGYGKDW GKYLN NLE DYLPL - WS YN FDPW T TF A0 2 00 Fzd GSISG 280 GSIYF 546 CARVMLIT 942 RSSQSLL 1138 LGS 1237 CMQG 1289 1S 1 NNYY TGGT DAFDIW HSNGYN NRA THWP - WG YYN YLD S YTF E0 2 00 Fzd GAISG 266 GSIYY 548 CARIGIAVA 882 RASQSV 1110 DAS 1188 CMQA 1284 1S 1 TSYF TGNT APVDHW GTYLT NRA TQFPL - WG YYN T TF G 02 00 Fzd GSISS 285 GYIYY 589 CARATYGG 760 TRSSSNI 1161 GNS 1220 CGTW 1267 1S 1 SSYY SGSTY DAFDIW GAGYDV IRPS DSSLS - WG YN H AWVF H0 2 00 Fzd GSISS 284 GYIYY 589 CARVRDYY 946 RASRSISS 1128 AAS 1175 CQQA 1314 1S 1 GGYY SGSTY DSSGYYYD YFN SLQ DTFPP - WS YN YFDYW S TF A0 3 00 Fzd ASFS 158 GEID 487 CARGGQG 835 SGDKVG 1154 EDS 1199 CQAW 1301 1S 1 GHY HTGS GYDWGHY HKYAS QRP DSSTD - WT TNYE HGLDVW S VVF B0 3 00 Fzd RAFT 329 ATISG 466 CAAASSLTS 678 1S 5 DNV GGGS TPYDLW - MA TFDD H0 8 00 Fzd RSFRT 333 AAIS 422 CNTVTYTG 1005 1S 5 NALG WTG GSYKNYW - GSTY A0 YA 9 00 Fzd SIDSI 356 AALTS 428 CNVITIVRG 1006 1S 5 NAM GGITY MGPRAYW - A HA B0 9 00 Fzd SIFSIN 357 ATIQS 465 CNVITIVRG 1006 1S 5 AMG GGRT MGPRAYW - NYA C0 9 00 Fzd YTFTS 391 GWIS 570 CARDGTPF 772 QGDSLRT 1052 GKN 1219 CNSRD 1300 1S 8 YGIS AYNG YSGSYYGS YYAS NRP NSGK - NTNY W S HKVF C0 A 7 00 Fzd GTFSS 295 GRIIPI 529 CARVPTSP 944 RSSQSLL 1136 FGS 1206 CMQN 1291 1S 8 YAIS LGIAN YDILTGPFD HSNGFN YRA LQTP - YA YW YVD S WTF D0 7 00 Fzd ASVSS 159 YRSK 542 CARWKNY 953 RASQGIR 1070 AAS 1177 CLQDY 1273 1S 8 NSAA WYN FDPW SDLA TLES SYPRT - WN DYA F E0 7 00 Fzd FTFSS 228 STISG 646 CAKDLVP 704 RASQSVS 1121 GAS 1213 CQQY 1410 1S 8 YAMS GGGS WGSSAFNI SYLA SRA GSSPP - TYYA W T TF H0 7 00 Fzd FTFST 243 SGVS 618 CARGQSEK 856 RASQGIS 1076 AAS 1165 CQQT 1394 4S 5 YEMN WNG WWSGLYG SALA ALQ YSTPR - SRTH MDVW S TF E0 YV 5 00 Fzd GTFST 298 GWIN 565 CWTGLLW 1031 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS SGNG FGESTDAF SYLN SLQ YSTPL - NTKY DIW S TF E0 S 3 00 Fzd GTFTY 307 GGIIPI 501 CASSMVR 964 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 RYLH FGTG VPYYYGM SYLN SLQ YSTPL - NYA DVW S TF G 06 00 Fzd GPFN 272 AGISR 445 CASKTTINS 958 1S 8 LFAM TGGN GWSREYH - G TGYA YW D0 9 00 Fzd GPFN 272 AGISR 445 CASKTTINS 958 1S 8 LFAM TGGN GWSREYH - G TGYA YW E0 9 00 Fzd GFFSS 268 AAISR 409 CNALAPGV 987 1S 8 FTMG NGVY RGSW - TRFA F0 9 00 Fzd SLFRL 360 ATIST 467 CTDEESW 1011 1S 8 NGM RGTT - G HYA G 09 00 Fzd GPFN 273 AGISR 445 CASKTTINS 958 1S 8 LLAM TGGN GWSREYH - G TGYA YW H0 9 00 Fzd SVVN 364 AAITS 425 CNRVGSRE 1001 1S 8 FVVM GGST YSYW - G NYA A1 0 00 Fzd AAIG 352 YKVK 404 CNAVTYN 994 1S 8 RTSDL WNG GYTIW - YTMG ERTYY B1 L 0 00 Fzd SIFSS 359 ALITT 455 CNAGAPA 986 1S 1 NTIY SGNT WTYRMGT - NYA YYPQFGS G W 12 00 Fzd STFST 362 AAISG 408 CVKFGMN 1028 2S 1 YAM SGEN LGYSGYDY - G TYYA W A0 1 00 Fzd STFSN 361 AAIS 411 CAAGPIAR 681 2S 1 YAM WGG WYRGDM - G GSTFY DYW B0 S 1 00 Fzd RMFS 331 AAISS 410 CAAGPIAR 681 2S 1 NYA GGSG WYRGDM - MG TYYS DYW C0 1 00 Fzd RTDG 337 ATVT 469 CAAGPIAR 681 2S 1 GYVM WRTG WYRGDM - G TTYY DYW D0 A 1 00 Fzd RTFSS 345 AAIS 421 CATLTPYG 974 2S 1 AAM WSGS TVASY - G TAYY E0 A 1 00 Fzd RTFSS 347 AAVN 430 CAAVFLSR 689 2S 1 YAM WSG NYEIQEYY - G GSTY RYQ F0 YA 1 00 Fzd RTFSS 347 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G GSTY DYW G YA 01 00 Fzd RSFST 336 TVISG 676 CAAGPIAR 681 2S 1 YPMG SGGS WYRGDM - TYYS DYW H0 1 00 2S Fzd RRFTT 332 AAVT 436 CYLEGPLD 1032 - 1 YGM WRSG VYW A0 G STYYA 2 00 Fzd RTFN 341 AAIS 415 CAKLGGSS 724 2S 1 RHV WSG WLREYDY - MG DSTY W B0 YA 2 00 Fzd RTFR 342 SAIS 603 CAAGPIAR 681 2S 1 AYAM WSG WYRGDM - G GSTY DYW C0 YA 2 00 Fzd RTFSE 343 AAIS 417 CNADSLRG 984 2S 1 YAM WSG IDYW - G GSTH D0 YA 2 00 Fzd FTFRE 199 SGISR 613 CAPRVLVT 734 2S 1 YAMT DGGR APSGGMD - TSYS YW E0 2 00 Fzd GDFT 267 AAVN 429 CAAVFLSR 689 2S 1 NYA WRG NYEIQEYY - MA DGTY RYQ F0 YS 2 00 Fzd RTFG 340 AAISY 424 CAAGPIAR 681 2S 1 TWA NGFS WYRGDM - MG TYYS DYW G 02 00 Fzd RTFSS 347 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G GSTY DYW H0 YA 2 00 Fzd RTFGS 339 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G GSTY DYW D0 YA 3 00 Fzd SIFSIY 358 AVVA 481 CNMRGN 1000 2S 1 AMG TGGA WYREGRP - TNYA AEFLSW E0 3 00 Fzd RTSSS 353 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G GSTY DYW F0 YA 3 00 Fzd RTFGS 339 AAIS 418 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G GSTY DYW G YA 03 00 Fzd QTFT 327 AAIS 420 CNAWVLV 996 2S 1 AYAM WSGS AGSRGTSA - G ATHY DYW H0 A 3 00 Fzd RTFSS 347 AAIS 419 CAAGPIAR 681 2S 1 YAM WSGR WYRGDM - G STYYA DYW A0 4 00 Fzd RTFSS 347 AAIS 418 CAAGPNYS 682 2S 1 YAM WSG WFMPSSS - G GSTY RLIW B0 YA 4 00 Fzd RRFTT 332 AAVT 435 CSADKLDY 1010 2S 1 YGM WRA LDDQPFKT - G GSTY WDYW C0 YA 4 00 Fzd GTSST 309 AAIN 405 CAAVFLSR 689 2S 1 YAM RSGG NYEIQEYY - G STYYA RYQ D0 4 00 Fzd GTFST 300 AAIS 416 CAAGPIAR 681 2S 1 YAM WSG WYRGDM - G DSTY DYW E0 YL 4 00 Fzd GTFSS 295 GWIS 577 CARGGAG 826 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS TYNG RFGEGMD SYLN SLQ YSTPL - ATNY VW S TF H0 A 4 00 Fzd YTFTS 391 GWIS 577 CASSKEKA 963 GLSSGSV 1035 YTN 1263 CLLYL 1271 1S 5 YGIS AYNG TYYYGMD STNYYPS TRS GRGI - NTNY VW S WVF A0 A 4 00 Fzd GTFSS 295 GRIIPI 529 CARLDPGY 886 TGTSSDV 1159 DVT 1196 CFSYA 1266 1S 5 YAIS LGIAN YYGMDVW GGYNSV KRP GSRF - YA S S D0 3 00 Fzd GTFSS 295 GGIIPI 499 CARVIFSTV 939 TRSSGSI 1160 END 1202 CQSYD 1430 1S 5 YAIS FGTA TTTNDIW ASNYVQ KRP YDHR - NYA S WVF F0 3 00 Fzd YTFSG 374 GTVT 549 CARVDGSG 933 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YYLH PILGT YYGIDYW SYLN SLQ YSTPL - ANYA S TF E0 4 00 Fzd GSFS 278 GRIIPI 530 CARTYLKA 930 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 NYAIS LGSA FDIW SYLN SLQ YSTPL - NYA S TF A0 6 00 Fzd YTFTN 383 GRIN 537 CARDRFDN 788 RASQGIS 1076 AAS 1179 CQQS 1351 4S 5 NFMH PNSG WFDPW SALA TLQ YNTP - GTNY S WTF F0 A 4 00 Fzd GTFSS 295 GRIIPI 529 CAREGRSR 808 RSSQSLL 1139 MG 1238 CMHG 1279 1S 5 YAIS LGIAN VYGGNSFD RRNGHN SNR LHPPF - YA YW YVD AP TF C0 3 00 Fzd YIFTD 368 GGIIPI 499 CARMSSDY 895 RASQGIS 1072 GAS 1215 CQQA 1312 3S 1 YYMH FGTA YDSSGYYR NNLN TLQ DSFPP - NYA RGMDVW S TF A0 1 00 Fzd YIFTD 368 GGIIPI 499 CARAWKG 761 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 YYMH FGTA LWFGEGTF SYLN SLQ YSTPL - NYA DYW S TF E0 1 00 Fzd GTFSS 295 GWIN 558 CARLAFDI 885 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 YAIS AGNG W SYLN SLQ YSTPL - NTTY S TF F0 A 1 00 Fzd YTFTG 379 GWIN 558 CAKDRGN 707 RASQGIS 1074 EVS 1204 CQQS 1370 3S 1 YYMH AGNG YGDYLDY NYLA SVQ YSTPL - NTTY W G AF A0 A 2 00 Fzd FTFSN 214 ALISY 454 CTRGSRIG 1015 RASQSIG 1084 AAS 1171 CQQG 1325 3S 1 SDM DGSH WFDPW RWLA RLQ FNFPL - N TYYA S TF C0 2 00 Fzd GTFSS 296 GGIIPI 505 CARARGG 749 RASQGIS 1072 TAS 1258 CLQDY 1274 3S 1 YTIS SGKT DSPLSL NNLN SLQ SYPYT - DYA S F E0 2 00 Fzd GTFRS 292 GGIIPI 499 CARGGWR 840 RASQSVS 1115 GAS 1217 CQQY 1405 3S 1 YAIN FGTA PDYYGSGS SDLA TRA ETWP - NYA YYSFDYW T VLTF F0 2 00 Fzd FTFGT 196 SGITG 616 CARMKDW 894 RASESVS 1056 GAS 1217 CQQY 1420 3S 1 YWVT SGGR FGAFDIW SSSFA TRA NNWP - TFYA T PNYTF G 02 00 Fzd FTFSR 220 SYISG 658 CARGLVIA 849 QANQDI 1038 AAS 1175 CQQT 1389 3S 1 YAMS DSGY TNWFDPW SNYLN SLQ YNPPR - TNYA S TF C0 3 00 Fzd YTFTS 392 GWIN 567 CAESLTSTA 691 RASQGIS 1072 AAS 1174 CQQS 1368 3S 1 YYMH TYNG DW NNLN SLQ YSTPF - NTNY R TF D0 P 3 00 Fzd YIFTD 368 NPTT 586 CARNVEGA 898 RASQGIS 1072 SAS 1252 CQQS 1364 3S 1 YYMH GNTG TSFPEFDY NNLN NLQ YSPPP - YA W S YTF E0 3 00 Fzd GTFSS 295 GGIIPI 499 CAKDIGSS 701 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 YAIS FGTA WYYYMDV SYLN SLQ YSTPL - NYA W S TF H0 3 00 Fzd FTFGT 196 SGITG 616 CARMKDW 894 RTSERSSI 1148 GAS 1217 CQQY 1420 3S 1 YWVT SGGR FGAFDIW SSFA TRA NNWP - TFYA T RNYTF A0 4 00 Fzd FAVSS 168 ASIW 463 CAPNESGN 733 RASQGIS 1072 KAS 1225 CQQS 1339 3S 1 SYMS FDGS VDYW NNLN SLE YSTPH - NQDY N TF C0 A 4 00 Fzd FTFSS 227 SAISG 600 CARDHGSS 774 QASQDI 1042 DVS 1195 CQHL 1304 3S 1 YAM SGGS WYQNTDA GNYLN NLE NSYPP - H TYYA FDIW R GDTF D0 4 00 Fzd FRFIS 177 GRVIP 545 CASSSDYG 966 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 HPIH ILGVT DYLKEPNY SYLN SLQ YSTPL - NYA GMDVW S TF G 04 00 Fzd FTFSN 216 SAIGT 595 CATAYRRP 969 RSSQSLL 1134 LGS 1237 CMQN 1293 3S 2 YAMT GGGT GGLDVW HSDGKTY NRA THWP - YYA LY S LTR D0 5 00 Fzd FTFSS 236 GRIKS 535 CARGSSSW 863 KSSQSLL 1036 LGS 1237 CMQN 1293 3S 2 YTMS KANG YDW HSDGKTY NRA THWP - GTTD LY S LTR E0 YA 5 00 Fzd FTFA 188 SYISS 659 CARGTFD 865 RASQGIS 1072 AAS 1170 CQQS 1372 3S 2 DYG GSYTI WLLSPSYD NNLN RLES YSTPL - MH YYS YW TF A0 6 00 Fzd FTFSN 217 SAISN 601 CTSSFLTGS 1018 RASQDIS 1065 AAS 1175 CQQS 1353 3S 2 YGM SGGS QPSGYW SYLA SLQ YRTPL - H TYYA S TF C0 6 00 Fzd FTFSD 207 SSTSG 642 CARHNPGY 877 RASQSVS 1116 DAS 1188 CQHR 1307 3S 2 YGM SGGN MGYYYGM SNLA NRA TSWPL - H SKYS DVW T TF G 06 00 Fzd GTFSS 296 GLVD 520 CTILPAAAA 1012 RASQRV 1083 DAS 1184 CQQY 1415 3S 2 YTIS PEDG GTYYYYG GNNLA IRAT KDWP - ETIYA MDVW TF H0 6 00 Fzd FTFSD 205 SSITR 639 CARDGGY 768 RASQSV 1109 GSS 1221 CQQY 1414 3S 2 HYMS TPSG W GSYLA NRA GTSLL - GTTE A TF B0 YA 7 00 Fzd YTFTN 383 GIINP 513 RRYCSSTSC 759 QASQGIS 1049 LGS 1233 CQQS 1368 3S 2 NFMH SGGS YPRDAFDI NNLN DRA YSTPF - TSYA W S TF D0 7 00 Fzd YTFTN 383 GWIN 563 CARSVGEV 926 RASQSIS 1098 AAS 1175 CQQS 1368 3S 2 NFMH PNSG GATMLGIG SYLN SLQ YSTPF - GTKY VWYWFDP S TF E0 A W 7 00 Fzd FTFSN 216 SAIGT 595 CATAYRRP 969 RSSQSLL 1134 LGF 1232 CMQN 1293 3S 2 YAMT GGGT GGLDVW HSDGKTY NRA THWP - YYA LY S LTR A0 8 00 Fzd LTVST 324 AGIG 440 CARDLVAA 782 RASQGIR 1069 GAS 1214 CQQS 1374 3S 2 NFMS WDST RPSNWDY NDLG TLQ YSTPR - NIGY W R VTF C0 A 8 00 Fzd FTFR 201 STISG 647 CARGGGYS 829 RSSRSLL 1143 LGS 1237 CMQS 1298 3S 2 NSAM SGGS SSW HSDGKTY NRA SHWP - H TYYS LY S KTF E0 8 00 Fzd FTFD 194 SAIGA 593 CASPTVTR 960 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 HNP GGGT R SYLN SLQ YSTPL - MN YYA S TF G 09 00 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YAIS AGNG GSYPDW SYLN SLQ YSTPL - NTTY S TF C1 A 0 00 Fzd FNFGI 172 SYISG 658 CARVGPG 936 RASQGIS 1076 AAS 1167 CQQT 1396 3S 4 YSMT DSGY GWFDPW SYLA NLL YSTP - TNYA G WTF D1 0 00 Fzd FTFSS 227 AGISA 442 CARPSTTG 901 RASQSIG 1085 AAS 1178 CQQS 1380 3S 4 YAM SGGS TKAFDIW SNLD TLET YSVPD - H TYYA TF E1 0 00 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 RASQSIS 1103 ZAS 1264 CQQS 1372 3S 4 YAIS AGNG GSYPDW ZYZN SLQ YSTPL - NTTY S TF A1 A 1 00 Fzd GTFSS 295 GRIIPI 528 CARGARLD 820 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YAIS FGTV YW SYLN SLQ YSTPL - NYA S TF G 11 00 Fzd YTFTG 379 GGIIPI 502 CASTDPSS 967 RASQSIG 1085 DAS 1189 CQQSF 1341 3S 4 YYMH FGTP GLDYW SNLD SLES IMPLT - HYA F H1 1 00 Fzd GTFSS 295 GWIN 564 CARGGSSD 838 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YAIS PNSG VR SYLN SLQ YSTPL - GTNY S TF C1 A 2 00 Fzd FTFSS 227 SVIST 652 CARGGSSD 838 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YAM SGDT VR SYLN SLQ YSTPL - H VLYT S TF F1 2 00 Fzd GTFSS 295 GIINP 513 CAKDGVV 698 RAIQSISS 1054 AAS 1175 CQQS 1372 4S 4 YAIS SGGS R YLN SLQ YSTPL - TSYA S TF B0 1 00 Fzd FTFSN 213 STISS 650 CARASRID 754 RASQDIR 1062 AAS 1179 CQQA 1311 4S 4 HYTS SGGR GGWPIIDH DELA TLQ DSFPL - TFYA L S TF C0 1 00 Fzd FTFTN 248 SAISG 600 CARATGFG 757 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YAMS SGGS TVVFDYW SYLN SLQ YSTPL - TYYA S TF D0 1 00 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 ZACLRIIS 1163 FAS 1205 CQQS 1372 4S 4 YAIS AGNG GSYPDW YLN SLQ YSTPL - NTTY S TF E0 A 1 00 Fzd GTFSS 295 GWIN 558 CARDGVE 773 RASQGIS 1073 DAS 1190 CQQS 1344 4F 4 YAIS AGNG NWLA SLQ HITPY - NTTY S TF F0 A 1 00 Fzd FTFSN 215 ALMS 456 CAKGIVGD 717 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YAM PDGTI YGAFDIW SYLN SLQ YSTPL - H IYYA S TF H0 1 00 Fzd FTFSS 230 SSINN 630 CAKDHLAV 700 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YGM SSRTV ADAHGR SYLN SLQ YSTPL - H FYA S TF B0 2 00 Fzd FTFSS 227 AVISY 474 CAGGEVYE 692 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YAM DGSN L SYLN SLQ YSTPL - H EYYA S TF E0 2 00 Fzd FTFST 242 AVISS 473 CAAPDVVV 685 RASQGIS 1076 AAS 1179 CQQA 1322 4S 4 YAM DGNN TADGYYW SALA TLQ NTVPF - H KYYT S TF F0 2 00 Fzd FTFA 190 ALISY 453 CAKTLVTS 728 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 NYA DGGT HALHIW SYLN SLQ YSTPL - MN KYYA S TF G 02 00 Fzd FTFA 189 ALISY 452 CAKTLVTS 728 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 NYA DGGN HALHIW SYLN SLQ YSTPL - MH KYYA S TF H0 2 00 Fzd GSFS 276 GEIN 489 CARGRRLV 858 TGSSSNI 1158 SDR 1255 CQSYD 1429 1S 5 GYYW HSGS RFTVTSAF GAGFGV NRP SSLRA - H TNYN DIW H S SVF E0 3 00 Fzd GTFSS 295 GGIIPI 504 CARIPKPR 883 RSSQSLL 1137 LGS 1234 CMQS 1297 5 YAIS LGIAN GYSYGDN HSNGNT DRT LQTPY - YA GSW YLD S TF B0 5 00 Fzd GNFK 271 GRIIP 525 CARQYCSG 908 RASQDIR 1063 QAS 1245 CQQS 1361 4S 6 NYGIT ALGT GSCYPDAF SALA SLIS YSMP - ANYA DIR QTF A0 7 00 Fzd FTFSS 233 GVISK 553 CASSRDGY 965 QASQDIR 1043 AAS 1175 CQQSS 1347 4S 6 YSMN DGDN NRLAFDIW NYLN SLQ RFWT - KYYA S F B0 7 00 Fzd GTFSS 295 GRIIPI 529 CARDGGD 767 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS LGIAN YGMDVW SYLN SLQ YSTPL - YA S TF A0 8 00 Fzd YTFTN 383 PNSG 537 CASQNYYG 961 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NFMH GTNY SGSYPGFD SYLN SLQ YSTPL - A YW S TF B0 8 00 Fzd YTFTY 394 GGIIPI 499 CATHDSSG 973 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 RYLH FGTA YYSFDYW SYLN SLQ YSTPL - NYA S TF D0 8 00 Fzd FSVSS 187 SAIGT 595 CTTRTYDSS 1024 RSSRSLL 1144 LGS 1237 CVQTT 1434 4S 6 NYM GGGT GYYETQNY HSNGNT NRA QSPLT - N YYA YMDVW YLQ S F E0 8 00 Fzd FTFSD 208 AAISY 423 CARSAVAG 916 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYMS DESN AFDIW SYLN SLQ YSTPL - KFYA S TF G 08 00 Fzd FTFRD 198 SGIS 615 CARRSGYS 913 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAM WNS GSVYYYYG SYLN SLQ YSTPL - N GSIGY MDVW S TF A0 A 9 00 Fzd FTFSS 221 AGIN 441 CARGPSHQ 854 RASQGIS 1076 AAS 1175 CQQS 1357 4S 6 FGMH WNG HTFDIW SALA SLQ YSHTA - GSVV S FTF B0 YA 9 00 Fzd YTFTN 383 GGFD 492 CARVGRGY 937 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NFMH PEDG SFDYW SYLN SLQ YSTPL - ETIYA S TF C0 9 00 Fzd DTFS 163 GRISA 538 CARSSGYV 924 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NYVIS YNGY GWFDPW SYLN SLQ YSTPL - KSYA S TF E0 9 00 Fzd FTFSN 218 SYISG 657 CARLPRRS 888 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYTS AGGS GKGSAFDI SYLN SLQ YSTPL - TEYA W S TF F0 9 00 Fzd GTFSS 296 NPNS 583 CARVGATS 935 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YTIS GNTG AGGMDV SYLN SLQ YSTPL - YA W S TF H0 9 00 Fzd YIFTD 368 GLVD 520 CAHSDFFS 693 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYMH PEDG GLSFGDW SYLN SLQ YSTPL - ETIYA S TF C1 0 00 Fzd FTFSN 214 SSIST 637 CARGSYW 864 RASQNIN 1081 RAS 1249 CQQY 1425 4S 6 SDM SGGS NYLA TLQ SSYPY - N TYYA S TI D1 0 00 Fzd TTLNK 365 GRITP 539 CALSSSWY 731 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS VVGV GGFDYW SYLN SLQ YSTPL - TNYA S TF E1 0 00 Fzd GFTFS 269 ALVG 458 CNTGIPML 1003 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 DHY YDGS YW SYLN SLQ YSTPL - QQFY S TF F1 G 0 00 Fzd FTFSD 208 SAISG 599 CARVSRGF 948 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYMS SGFTY AFDYW SYLN SLQ YSTPL - YA S TF G 10 00 Fzd GTFSS 295 GRIIPI 529 CARESVNN 813 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS LGIAN YYYMDVW SYLN SLQ YSTPL - YA S TF A1 1 00 Fzd FTFSS 227 ALTSY 457 CAKTGRGY 726 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAM DGSK AFDIW SYLN SLQ YSTPL - H KFYA S TF C1 1 00 Fzd FTFSS 232 KANG 535 CAKAGQQ 696 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YNM GTTD LDW SYLN SLQ YSTPL - N YA S TF E1 1 00 Fzd FTFTS 249 GGIIPI 499 CATVQTNY 977 RASQSIS 1094 AAS 1175 CQQY 1426 4S 6 SAM FGTA YDSSGRFS RWLA SLQ VSYPL - Q NYA YRAHYFDY S TF H1 W 1 00 Fzd YTFTN 383 GRIN 537 CARGQGYS 855 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NFMH PNSG SGWYRGD SYLN SLQ YSTPL - GTNY AFDIW S TF A1 A 2 00 Fzd FAFD 165 KAYG 490 CAKDRGYS 708 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 DYA GTTE SGWYLDY SYLN SLQ YSTPL - MH YA W S TF D1 2 00 Fzd FNFSS 173 SVIYG 653 CARGGSG 836 RASQSIS 1098 AAS 1175 CQQS 1372 5S 7 YTMR GGNT GNLSYW SYLN SLQ YSTPL - NYA S TF H0 1 00 Fzd GTFSS 295 GMIIP 521 CTRPYDAF 1016 RASQSIS 1098 AAS 1175 CQQS 1372 5S 7 YAIS FLGIT DIW SYLN SLQ YSTPL - NYA S TF A0 2 00 Fzd YTFAS 373 GWIN 558 CARLSVWK 890 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YGM AGNG WEQVTN SYLN SLQ YSTPL - H NTTY WFDPW S TF C0 A 2 00 Fzd GTFTS 305 GWIN 557 CTTGLFPYY 1022 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS AGNG RYNWNND SYLN SLQ YSTPL - NTKY AFDIW S TF E0 S 2 00 Fzd GTFSS 295 GWM 583 CAKWHIG 729 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS NPNS ATGNWFD SYLN SLQ YSTPL - GNTG PW S TF A0 YA 3 00 Fzd YTFTN 383 GGIFP 494 CARDRPTS 792 RASQGIS 1072 DAS 1193 CQQSF 1342 5S 8 NFMH IYGIS SWYAFDY NNLN TLQ SAPITF - TYA T H0 3 00 Fzd FSFSS 181 SYISS 670 CARYGDYG 954 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 TAMS SGSIT DYW SYLN SLQ YSTPL - HYA S TF F0 4 00 Fzd YTFTN 383 GWIN 558 CARVATGN 932 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 NFMH AGNG AFDIW SYLN SLQ YSTPL - NTTY S TF H0 A 4 00 Fzd FTFSS 239 AGISG 444 CARGGLLF 831 QASQDIS 1046 KAS 1226 CQQS 1373 5S 8 YWM SGKT DYW NYLN SLES YSTPR - H TFYA TF B0 5 00 Fzd FTFTS 251 GWM 583 CARRTAVA 914 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 SAVQ NPNS GTIDYW SYLN SLQ YSTPL - GNTG S TF F0 YA 5 00 Fzd GTFSS 295 GWIS 573 CARGGWT 841 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS PYNG NYGGNLDY SYLN SLQ YSTPL - NTNY W S TF G A 05 00 Fzd YTFTS 392 GRIN 537 CARVPDF 943 RASQGIS 1075 AAS 1175 CQQT 1392 5S 8 YYMH PNSG WSGYLDY RTLZ SLQ YSMPI - GTNY W S TF H0 A 5 00 Fzd YTFTY 394 GGIIPI 499 CARDSYPY 800 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 RYLH FGTA GMDVW SYLN SLQ YSTPL - NYA S TF D0 6 00 Fzd GTFSS 295 GRVIP 545 CAREYLGS 815 RASQSV 1108 GAS 1213 CQQY 1409 5S 8 YAIS ILGVT FDIW GSNLA SRA GSSPP - NYA T FTF F0 6 00 Fzd FTFTG 247 GGILP 509 CARGARLY 822 RASQSVS 1114 GAS 1217 CQQR 1335 5S 9 SAVQ IYGTT GFDYW RNLA TRA SNWPI - KYA T TF A0 7 00 Fzd FTFTS 251 GWM 583 CARGRGQ 857 RASQGIS 1076 GAS 1215 CLQDY 1272 5S 9 SAVQ NPNS QWLTGYY SALA TLQ NYPFT - GNTG GMDVW S F B0 YA 7 00 Fzd FTFSS 233 SYIEN 654 CARAPYYY 748 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YSMN DGSIT GSGSLFRL SYLN SLQ YSTPL - TYA DYW S TF C0 7 00 Fzd GTFN 291 GGIIPI 499 CARAGSGY 740 RASQSIN 1090 AAS 1175 CQQT 1388 5S 9 SYAIA FGTA YNFDYW RWLA SLQ YNIPIT - NYA S F D0 7 00 Fzd FSFSS 182 AYINS 483 CAKTKLPI 727 RASQSIN 1089 AAS 1176 CQQY 1402 5S 9 YGM RGSL W RNYLG SRV DSWP - H MYYA T PTF F0 7 00 Fzd GSFS 274 GGIIPI 499 CATGYYYD 972 RASQGIS 1072 AAS 1175 CQHY 1309 5S 9 GYAI FGTA YYFDYW NNLN SLQ YNLPL - N NYA S TF G 07 00 Fzd GTFT 303 GLVD 520 CARTYRIV 931 RASQTIN 1125 KAS 1224 CQQA 1318 5S 9 NNF PEDG GATPRYYY NQLA NLE NSFPV - MH ETIYA YGMDVW T TF H0 7 00 Fzd YIFTD 368 GWIN 562 CARGPRDS 853 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YYMH PNSG GYYPGGAF SYLN SLQ YSTPL - GTIYA DIW S TF B0 8 00 Fzd FAFSS 166 SAIDG 592 CARDRQLG 794 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 HWM SGGS WAHWYFD SYLN SLQ YSTPL - H TYYA LW S TF D0 8 00 Fzd YTFTG 379 GWIN 558 CARDRDY 787 QTSQDIN 1053 KAS 1226 CQQS 1366 5S 9 YYMH AGNG W NNLN SLES YSSPP - NTTY TF G A 08 00 Fzd FTFSS 230 SAIGT 595 CALLVGAA 730 QASQDIS 1046 AAS 1179 CLQHK 1276 5S 9 YGM GGGT RGISYYYYY NYLN TLQ SFPTF - H YYA GMDVW S C0 9 00 Fzd YTFTS 389 GWIN 558 CARDRPYS 793 RASQSVS 1117 GAS 1217 CQQR 1339 5S 9 YAM AGNG SGWYYPAF SNQLA TRA YNWP - H NTTY DIW T PSITF D0 A 9 00 Fzd FNLR 175 SRISN 627 CARDADSS 762 RASQSVS 1116 DAS 1188 CQQR 1334 5S 9 RYNM SGSLV GYYRYDAF SNLA NRA NNWL - N YYA DIW T YTF E0 9 00 Fzd YTFTD 376 GIINP 513 CARHVYGS 878 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YYMH SGGS GTYNNWF SYLN SLQ YSTPL - TSYA DPW S TF A1 0 00 Fzd YTFTS 392 GWM 583 CARGGPIH 834 RASQGIS 1072 AAS 1179 CQQT 1385 5S 9 YYMH SPNS YYYYYYMD NNLN TLQ NLFPY - ANTG VW S TF D1 YA 0 00 Fzd GAFS 265 GRIIP 534 CAKGGWR 715 RASQSVS 1116 GAS 1217 CQQY 1421 5S 9 TSSIS VLGT SSFDPW SNLA TRA NSWP - ANYA T LTF H1 0 00 Fzd YTFTS 390 GGFD 492 CAKAGDW 695 RASQSIS 1094 AAS 1175 CQQT 1386 5S 9 YDIN PEDG GLYGMDV RWLA SLQ NTFPF - ETIYA W S TF B1 1 00 Fzd FTFTG 247 GGILP 509 CARGARLY 822 RASQSVS 1113 GAS 1217 CQQR 1335 5S 9 SAVQ IYGTT GFDYW RKLA TRA SNWPI - KYA T TF C1 1 00 Fzd YTFTN 383 GWIN 561 CAREANYD 806 RASQSLR 1106 GAS 1217 CQQY 1401 5S 9 NFMH PNSG ILTGYIRPD SKLA TRA ANSP - DTKF AFDIW T WTF D1 A 1 00 Fzd GTFSS 295 GWIN 557 CTTTEYSSS 1025 QASQDIS 1046 GAS 1215 CQQLS 1331 5S 9 YAIS AGNG PDYYYGM NYLN TLQ RYPSL - NTKY DVW S F E1 S 1 00 Fzd GTFT 304 GGIIPI 499 CARSSDLRI 922 RASQSVS 1116 GAS 1209 CQQY 1413 5S 9 RNSIS FGTA FDYW SNLA NRP GSSPY - NYA T TF G 11 00 Fzd YTFAS 372 GWIN 558 CARDGIW 769 RASQSIS 1098 AAS 1175 CQQS 1372 5S 10 YDIH AGNG DIFDYW SYLN SLQ YSTPL - NTTY S TF H1 A 1 00 Fzd YIFTD 368 GVIFP 551 CARGGSTG 839 RASQSV 1107 AAS 1175 CQQA 1321 5S 10 YYMH VYPT YYGMDVW GRWMA SLQ NTFPF - PDYA S TF E12 00 Fzd GTFSS 295 GRIVP 541 CARDTCSS 801 RASQSIS 1098 AAS 1175 CQQS 1372 5S 10 YAIS IVDV TSCSPDYW SYLN SLQ YSTPL - VKYA S TF F1 2 00 Fzd FTFSS 233 SAIGT 595 CAREGWF 810 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YSMN GGGT GESPFGM SYLN SLQ YSTPL - YYA DVW S TF A0 1 00 Fzd YTFTR 385 GWIS 576 CASPTGMT 959 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YAVH TFND TNFDYW SYLN SLQ YSTPL - NTNY S TF F0 A 1 00 Fzd YIFTD 368 GGIIPI 499 CAKGSYYY 723 RASQGIS 1072 AAS 1166 CQQT 1387 6S 10 YYMH FGTA DSSGYYW NNLN NLE SSTPL - NYA DAFDIW T TF H0 1 00 Fzd YIFTD 368 GGIIP 507 CARDITGA 775 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YYMH LFGTT DGMDVW SYLN SLQ YSTPL - DYA S TF A0 2 00 Fzd GTFSS 295 GRIIP 533 CARDVCSG 802 RASQGIS 1072 DAS 1189 CQQT 1390 6S 10 YAIS TVGT GSCSPDV NNLN SLES YNTPR - ANYA W TF D0 2 00 Fzd FTFTS 250 GGIIPI 499 CARDGSSG 770 RASQGIS 1072 AAS 1175 CLQH 1277 6S 10 SATQ FGTA WYSPNAF NNLN SLQ NGYPI - NYA DIW S TF E0 2 00 Fzd FTFR 200 SRISP 628 CARSPRW 920 RSSQSLL 1138 RVS 1251 CMQG 1288 6S 10 MYG DGRT YDAFDIW HSNGYN SRF THWP - MH TTYA YLD S PTF H0 2 00 Fzd YIFTD 368 GWIN 558 CARDPIMF 784 RASESVS 1055 GAS 1213 CQQY 1419 6S 10 YYMH AGNG GDQPGWF SNLA SRA NKSPS - NTTY DPW T F A0 A 3 00 Fzd GTFSS 295 GWIN 556 CAREGYDF 811 RASQTIS 1126 EVS 1203 CQQS 1378 6S 10 YAIS AGNG WSGPYAF RYLN SLQ YSTP - NTKY DIW G WTF B0 A 3 00 Fzd GTFSS 293 GGIIPI 499 CARGGYYY 843 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 NVIS FGTA GMDVW SYLN SLQ YSTPL - NYA S TF C0 3 01 Fzd YIFTD 368 GGIIPI 499 CARMSSDY 895 RASQGIS 1072 GAS 1215 CQQA 1312 4S 1 YYMH FGTA YDSSGYYR NNLN TLQ DSFPP - NYA RGMDVW S TF B0 1 01 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 RASQSIS 1096 AAS 1175 CQQS 1372 4S 4 YAIS AGNG GSYPDW SHZN SLQ YSTPL - NTTY S TF D0 A 1 01 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 RASQSIZ 1105 AAS 1175 CQQS 1372 4S 4 YAIS AGNG GSYPDW ZYZN SLQ YSTPL - NTTY S TF E0 A 1 01 Fzd GTFSS 295 GWM 581 CARHYYGS 879 RASQSIS 1098 AAS 1175 CQQS 1368 4S 4 YAIS NPNN GNYRDW SYLN SLQ YSTPF - GNTT S TF G YA 01 01 Fzd FTFSS 223 SGISG 608 CAKPGIAA 725 RASQGIS 1076 GAS 1218 CQQS 1373 4S 4 NAM SGGS AGTNNWF SALA TVE YSTPR - H TYYA DPW S TF A0 2 01 Fzd FTFSS 227 SGISG 611 CARPSTTSF 902 RASQSVS 1116 GAS 1217 CQQY 1403 4S 4 YAM SGSST GMDVW SNLA TRA DTPLR - H YYA T TF B0 2 01 Fzd YTFTS 392 PNSG 537 CARVPDF 943 RASQGIS 1076 AAS 1175 CQQT 1392 4S 5 YYMH GTNY WSGYLDY SALA SLQ YSMPI - A W S TF C0 2 01 Fzd GTFST 299 GIINP 513 CARAKGSG 744 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS SGGS WYVGSAF SYLN SLQ YSTPL - TSYA DIW S TF D0 2 01 Fzd FTFSD 206 GFIRS 490 CARATQEL 758 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 SYMS KAYG LLPYGMDV SYLN SLQ YSTPL - GTTE W S TF E0 YA 2 01 Fzd YTFTS 392 GRIN 537 CARVPDF 943 RASQGV 1079 AAS 1175 CQQT 1392 4S 5 YYMH PNSG WSGYLDY STZLS SLQ YSMPI - GTNY W S TF F0 A 2 01 Fzd YTFTS 392 GIISP 516 CARWGDY 951 RASQGIS 1076 ATS 1183 CQQV 1399 4S 6 YYMN SGGS GDLYYFDY SALA TLQ NSYPP - TSYA W S TF G 02 01 Fzd YIFTD 368 GRIN 537 CARARSSG 751 RASQSVS 1120 AAS 1180 CQQS 1376 4S 6 YYMH PNSG WTDAFDI SWLA TLQ YSTPT - GTNY W T F H0 A 2 01 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAIS AGNG GSYPDW SYLN SLQ YSTPL - NTTY S TF A0 A 3 01 Fzd FTFSS 232 GRIKS 535 CARAGDSP 739 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YNM KANG DYW SYLN SLQ YSTPL - N GTTD S TF B0 TA 3 01 Fzd GTFSS 295 GWIS 574 CARAMWS 745 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YAIS PYNG YGQQNAF SYLN SLQ YSTPL - YTKY DIW S TF E0 A 3 01 Fzd FTFTS 251 GWM 583 CARRTAVA 914 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 SAVQ NPNS GTIDYW SYLN SLQ YSTPL - GNTG S TF G YA 03 01 Fzd YTFTS 387 GRIN 537 CARVKWEL 940 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 SAIH PNSG AIDYW SYLN SLQ YSTPL - GTNY S TF H0 A 3 01 Fzd YIFTD 368 GWM 583 CARGGSRY 837 RASQGIS 1074 AAS 1175 CQQS 1368 4S 8 YYMH NPNS DFWSGHW NYLA SLQ YSTPF - GNTG YFDLW S TF B0 YA 4 01 Fzd YTFTG 379 GRIN 537 CARDVPKL 804 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YYMH PNSG VTRGVAYG SYLN SLQ YSTPL - GTNY MDVW S TF E0 A 4 01 Fzd YSFTT 371 GWIN 558 CARAAAGS 736 RASQGIS 1072 EAS 1197 CQQS 1381 4S 8 YGM AGNG YGGGYW NNLN SVA YTSTP - N NTTY S LNSF F0 A 4 01 Fzd FTFSS 231 SAISG 600 CARDLTPF 780 RASQSVS 1112 GAS 1216 CQQY 1423 4S 8 YGMS SGGS TQQQLVLG GYLA TRA NYWP - TYYA LL A PAF G 04 01 Fzd FTFTS 251 GRIVP 540 CARSGYNR 919 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 SAVQ AIGFT RGYFDYW SYLN SLQ YSTPL - QYA S TF H0 4 01 Fzd GTFSS 295 GGIIPI 499 CARVTLGA 949 RASQGIS 1072 DAS 1189 CLQH 1278 4S 8 YAIS FGTA SVDAFDIW NNLN SLES NSLPF - NYA TF A0 5 01 Fzd GTFSS 295 GWVS 587 CTTDRRYS 1021 RASQSVS 1116 GVS 1223 CQQY 1418 4S 8 YAIS PNTG TYFDLW SNLA NRA NIWP - NTVY T RTF B0 A 5 01 Fzd YTFAS 373 GWIN 558 CARLSVWK 890 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YGM AGNG WEQVTN SYLN SLQ YSTPL - H NTTY WFDPW S TF C0 A 5 01 Fzd GTFTS 305 GWIN 557 CTTGLFPYY 1022 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YAIS AGNG RYNWNND SYLN SLQ YSTPL - NTKY AFDIW S TF D0 S 5 01 Fzd FTFTG 247 GGILP 509 CARGARLY 821 RASQZVS 1127 GAS 1217 CQQR 1335 4S 9 SAVQ IYGTT GCDYW RZZA TRA SNWPI - KYA T TF F0 5 01 Fzd FTFSS 226 SAIGT 595 CARKVKGY 884 RVSQGIS 1151 AAS 1175 CQQTF 1384 4S 9 SWM GGGT CSGGSCYG SALA SLQ SVPW - H YYA YW S TF G 05 01 Fzd FTFSN 216 STISG 648 CARHGRIA 876 RASQSIS 1098 AAS 1175 CQQS 1372 4S 9 YAMT SGVS ADIW SYLN SLQ YSTPL - TFYA S TF H0 5 01 Fzd FTFZZ 254 GGILP 509 CARGARLY 822 RASQSVS 1114 GAS 1217 CQQR 1335 4S 9 SZVQ IYGTT GFDYW RNLA TRA SNWPI - KYA T TF A0 6 01 Fzd FTFSS 233 SYIEN 654 CARAPYYY 748 RASQSIS 1098 AAS 1175 CQQS 1372 4S 9 YSMN DGSIT GSGSLFRL SYLN SLQ YSTPL - TYA DYW S TF B0 6 01 Fzd FTFTG 247 GGILP 509 CARGARLY 822 RASQSVS 1114 GAS 1217 CQQR 1335 4S 10 SAVQ IYGTT GFDYW RNLA TRA SNWPI - KYA T TF C0 6 01 Fzd FTFSR 219 SGIGV 605 CARDAYN 763 RASQSIS 1095 AAS 1175 CQQR 1340 4S 10 YAM GGGT WFDPR RYLN SLQ YSTPL - H YYA S TF D0 6 01 Fzd YIFTD 368 GVIFP 551 CARGGSTG 839 RASQSV 1107 AAS 1175 CQQA 1321 4S 10 YYMH VYPT YYGMDVW GRWMA SLQ NTFPF - PDYA S TF F0 6 01 Fzd FTFSS 227 SAIGA 593 CARDAYN 764 RASQSIS 1098 AAS 1175 CQQS 1372 4S 10 YAM GGGT WFDPW SYLN SLQ YSTPL - H YYA S TF G 06 01 Fzd FTFSS 229 SAIGT 595 CARDAYN 764 RASQSIS 1098 AAS 1175 CQQS 1372 4S 10 YDM GGGT WFDPW SYLN SLQ YSTPL - N YYA S TF H0 6 01 Fzd FTFSN 210 SAIGT 595 CAREGSYY 809 RASQNIG 1080 GAS 1208 CQQY 1417 4S 10 AQM GGGT DWYFDLW SRLA NRA NHWP - S YYA S PLFTF A0 7 01 Fzd IIFSP 313 ALISS 450 CHFGVASV 980 7S 8 NDM GGST GLNYW - G SYA E0 8 01 Fzd RTFSS 346 AAVS 432 CNLAQRGE 998 7S 8 FVMG ASGG TYW - YTWY H0 A 8 01 Fzd LAFN 317 AAIS 414 CAAGFPTV 680 7S 8 GYTM WSD FVVDGEYD - G NTYY YW A0 A 9 01 Fzd FTLDY 255 ADITS 437 CNAVTYN 994 7S 8 YAIS GGST GYTIW - NYA B0 9 01 Fzd LTFSD 319 ASST 464 CNAVTYN 994 7S 8 YTVG GGGV GYTIW - FENY C0 A 9 01 Fzd RIFSS 330 PRIPS 590 CEVHNFGA 979 8S 4 YAQA DSTTF TYW - YA D0 6 01 Fzd RTFS 344 AVISR 472 CNAVSTD 992 8S 4 NYV SGGN WTTDYW - MG TYYT E0 6 01 Fzd RTFST 349 AAIS 414 CNSFPLRL 1002 8S 4 YGM WSD HDW - G NTYY F0 A 6 01 Fzd LAIDD 318 SYIST 673 CNAVTYN 993 8S 5 YYMV SDGS GYSIW - TYYA G 06 01 Fzd LAFN 317 AQIS 460 CNADYGT 985 8S 5 GYTM WTG WYGIGW - G GSTD H0 YA 6 01 Fzd LAFN 317 AAIS 412 CNMGLGY 999 85 5 GYTM WMS SEYRPLGY - G NTYY W A0 A 7 01 Fzd SAFS 355 AAIT 426 CNAVWKF 995 8S 5 NYA WSGA GTTHW - MG RTYY B0 A 7 01 Fzd LTIDD 323 SYISA 656 CNAVTYN 994 8S 7 YYVV GDGF GYTIW - TYYA C0 7 01 Fzd GSFS 277 GEIN 489 CARDLRFY 778 RSSRSLL 1147 LGS 1237 CMQG 1290 7S 4 GYYW HSGS SSSWRRVG HTSGYNY NRA TRWP - S TNYN MDVW LD S TF F0 9 01 Fzd YTITT 396 GWIN 555 CARGWTTI 872 RSSRSLL 1146 LGS 1237 CMQA 1281 7S 4 YAIH ADTG SSLGVW HTNGYN NRA LQTPL - DTAY YLD S TF G S 09 01 Fzd NIFRI 326 AALT 427 CNTVTYNA 1004 7S 5 YAIA GQRT GCYKKYW - TNYA H0 ASIT 9 01 Fzd LAFN 317 WNG 462 CNARLDAV 988 7S 5 GYTM RYTYY YGHSRYDS - G A W A1 0 01 Fzd NFFS 325 GAISR 484 CAAGVTGS 684 7S 5 NYPL TGSG WRYW - G TFYA B1 0 01 Fzd RSFSN 335 AVSW 479 CNAVTYN 994 7S 8 YRVA SVGM GYTIW - TYYA C1 0 01 Fzd GTFG 288 GLISR 518 CNAVNGRL 991 7S 8 SYAV NAGN NYW - G TLYA D1 0 01 Fzd RTFSS 348 AAVS 431 CAAPQSPN 687 7S 8 YSLA ASGA MYIRTDQL - NTYY WWYKYW E1 A 0 01 Fzd RSFST 336 TVISG 675 CAAGPTLP 683 8S 1 YPMG SGGS FRYW - TYYA D0 7 01 Fzd RAFS 328 AAIN 406 CNARLSFA 989 8S 1 NYA WSG GGMGYW - MG DSAY E0 YA 7 01 Fzd IKSMF 314 AFITR 438 CNAVSTD 992 8S 1 DMNF GGTT WTRDYW - MG RYG F0 7 01 Fzd LTIDD 322 SYIGT 655 CNAVTYN 994 8S 1 YYMV SDGT GYTIW - TYYA G 07 01 Fzd RVFSS 354 AGIAS 439 CKVHNFGA 983 8S 4 YAQA DSTTF TYW - YA H0 7 01 Fzd RIFSS 330 ASIPS 461 CKVHNFEA 982 8S 4 YAQA DGTT TYW - FYA A0 8 01 Fzd LTFST 321 AAIN 407 CNSFPLRL 1002 8S 4 YGM WSGR HDW - G STVY B0 A 8 01 Fzd RTLSS 351 ALISL 449 CNAVSTD 992 8S 4 YVVG SGAS WTTDYW - TYYA C0 8 01 Fzd IKSMF 314 AFITR 438 CNAVSTD 992 8S 5 DMNF GGTT WTRDYW - MG RYG D0 8 01 Fzd RTDG 338 GAIT 486 CNVLAQN 1007 8S 5 MQA WSLG DGDYRTYG - MG SAFY E0 A 8 01 Fzd RTFSS 346 AAVS 432 CNAVWKF 995 8S 5 FVMG ASGG GTTHW - YTWY F0 A 8 01 Fzd RTFSS 346 AAVS 432 CNAVCKFG 990 8S 5 FVMG ASGG TTHW - YTWY G A 08 01 Fzd RTFSS 346 AAVT 434 CNAVWKF 995 8S 5 FVMG ASGG GTTHW - YAWY H0 A 8 01 Fzd ITFSF 316 AVFIA 470 CNGVTYN 997 8S 8 NSVG GYGA GYTIW - YYA A0 9 01 Fzd HDFS 310 ATIS 468 CAAQKPYY 688 8S 8 STYG WGG NGHFYAD - VG TNIA DKHYDHW B0 9 01 Fzd ITFGF 315 AVFN 471 CNAVTYN 994 8S 8 DSVG AGYR GYTIW - AYYA C0 9 01 Fzd RTFS 350 AAVS 433 CNAVTYN 994 8S 8 WYS WSGV GYTIW - MG STYYP D0 9 01 Fzd ITFSF 316 AVFIA 470 CIGVTYNG 981 8S 8 NSVG GYGA YTIG - YYA E0 9 01 Fzd RTDG 338 GAIT 485 CNVLAQN 1008 8S 8 MQA WSLG DGDYRTY - MG IAFYA W F0 9 01 Fzd HDFS 311 AAIS 413 CAAQKPYY 688 8S 8 STYG WRGT NGHFYAD - VG NIA DKHYDHW G 09 02 Fzd DSVS 160 GRAY 524 CVRDLRPS 1029 RASQSIG 1087 YAS 1260 CHQS 1268 1S 8 SNSA YKSR GDLNFDY SSLH QSV GRVP - AWN WYYD W S VTF A0 YA 1 02 Fzd GSISS 283 GSIYH 547 CARFYYDIL 817 RSSRSLL 1142 TLS 1259 CMQSI 1295 1S 1 GGYS SGSTY NGYSYFDY DTDDGN HRA QLPW - WS YN W TYLD S TF C0 1 02 Fzd FTFSS 230 AVISY 475 CAKGSVFG 721 RSSQSLV 1140 KISN 1230 CMQA 1283 1S 1 YGM DGSN LKAGGYAD HSDGNT RFS TQFPH - H KYYA YW YLS TF D0 1 02 Fzd YTFTS 391 GWIS 570 CARDGTPF 772 QGDSLRT 1052 GKN 1219 CNSRD 1300 1S 8 YGIS AYNG YSGSYYGS YYAS NRP NSGK - NTNY W S HKVF E0 A 2 02 Fzd DSVS 162 GRTY 544 PRLDYW 1034 RSSQSLL 1133 MLS 1240 CMQR 1294 1S 8 SNSG YRSKY DSDDGN SRA LEFPY - AWN YNGY TYLD P TF G A 02 02 Fzd DSVS 160 GRTY 542 CARSQATG 921 RSSQNIF 1131 SAS 1254 CQQS 1349 1S 8 SNSA YRSK ERFDYW QSLN SLQ YNSPI - AWN WYN S TF A0 DYA 3 02 Fzd FTFSS 228 SVIST 652 CADGSGTS 690 RASQSIS 1098 AAS 1175 CQQS 1372 2S 4 YAMS SGGT HR SYLN SLQ YSTPL - VLYT S TF H0 6 02 Fzd YIFTD 368 GGIFP 493 CAKGSYYY 722 RASQGIS 1071 AAS 1166 CQQT 1391 2S 10 YYMH IFGTA DNSGYYW NNIN NLE YSIPFT - NYA DAFDIW T F A1 1 O GFTFS 270 VISGD 677 NFIKYVFA 1033 SGDKLGK 1152 EKD 1200 SSFAG 1435 M HYTLS GSYT N KYAS or or NRP or NSLE or P- YYAD SGDNIGS 1153 SG 1201 or 1436 18 SVKG FYVH or QSYA R5 DKS NTLSL NRP SG 02 Fzd FTFSS 228 SAISG 600 CAKGLWG 718 RASQSVS 1118 GAS 1212 CQQR 1337 7S 5 YAMS SGGS PLLNW SNYLS SRA TNWP - TYYA P PRVTF H0 2 02 Fzd DSVS 161 GRTY 543 CTRGNWN 1014 SGTSSNI 1157 GNN 1219 CSAW 1432 7S 8 SNSA YRSK VGLANW GAGYDV NRP DDNL - TWN WYSD H S NGVV B0 YA F 3 02 Fzd RSFSI 334 AAIS 418 CNVITIVRG 1006 7S 5 YNTA WSG MGPRAYW - GSTY E0 YA 1 00 Fzd LTFSI 320 SAISG 597 CARGVYPY 870 QASQDIS 1046 AAS 1175 CQQS 1371 4S 5 YAM DGAL SSKHKPSY NYLN SLQ YSTPL - H TYYA YYYGMDV S FTF D0 W 5 00 Fzd YDFTT 367 GGVI 511 CARGYYYG 874 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YGIH PAFG MDVW SYLN SLQ YSTPL - ATDY S TF D0 S 4 00 Fzd GTFSS 295 GWIN 558 CASGLGYF 957 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YAIS AGNG DYW SYLN SLQ YSTPL - NTTY S TF B0 A 5 00 Fzd YTFTN 383 GGIIPI 502 CARTLTTPP 929 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 NFMH FGTP YYYGMDV SYLN SLQ YSTPL - HYA W S TF G 03 00 Fzd FTFSN 214 SAIGT 594 CTRDLYGG 1013 KSSQSLL 1036 LGS 1237 CMQG 1286 4S 5 SDM GGDT YRDYW HSDGYTY NRA LQTP - N YYA LY S WTF F0 3 00 Fzd YIFTG 369 GRIN 537 CARGGEYS 827 RATQTIS 1129 AAS 1171 CQQY 1427 4S 5 YYMH PNSG SGWTYYYY TYLN RLQ YSYP - GTNY YGMDVW S WTS C0 A 4 00 Fzd YTFTY 394 GMIN 522 CARDVMD 803 RASQGIS 1072 AAS 1165 CQHL 1303 4S 5 RYLH PIGGS VW NNLN ALQ NNFPL - INYA S TF B0 6 00 Fzd FSVG 186 SSISS 634 CARGPKT 851 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 SNYM GNSYI MWEDRPD SYLN SLQ YSTPL - T YYA YW S TF F0 6 00 Fzd FTFST 245 GFIRS 491 CARLTGGA 892 RASQGIS 1072 GAS 1211 CQQS 1346 4S 5 YSMI KDYG VAGTHRDY NNLN SLQ HSSPR - GTTE W S TF A0 YA 4 00 Fzd FTFSS 237 SAIGT 595 CARGSSGY 862 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YVMS GGGT YVAW SYLN SLQ YSTPL - YYA S TF A0 5 00 Fzd FTFSN 212 AGVSI 447 CARDQND 785 RSSQSLL 1135 LGS 1235 CMQG 1285 4S 5 HYMS DANK SWYRSDY HSDGYTY HRA LQTPH - KYYA W LY S TF F0 5 00 Fzd GTFSS 295 GRIN 537 CARGSGYD 861 RASQSIS 1092 AAS 1175 CQQS 1350 3S 1 YAIS PNSG FFDYGMD NNLN SLQ YNTPF - GTNY VW S TF C0 A 1 00 Fzd DTFS 164 GLVD 520 CAKASTPM 697 RASQSIG 1085 AAS 1179 CQQN 1333 3S 1 NYVL PEDG VQGAPDY SNLD TLQ YATPR - S ETIYA W S TF H0 1 00 Fzd GTFN 289 GGIIPI 499 CATTQGVY 976 RASQSIS 1098 AAS 1175 CQQS 1372 3S 1 RYAIT FGTA SSSWYGG SYLN SLQ YSTPL - NYA GRAFDIW S TF H0 2 00 Fzd YTFTY 394 GRIN 537 CWGGSYY 1030 RASQGIS 1072 AAS 1175 CQQA 1316 3S 1 RYLH PNSG GDYW NNLN SLQ NSFPI - GTNY S TF H0 A 4 00 Fzd FTFSS 227 SSISW 638 CARGSGIA 860 TSSQSLL 1162 LGS 1237 CMQG 1289 3S 2 YAM NSGR ASGSYW HSDGKTY NRA THWP - H VDYA LY S YTF A0 5 00 Fzd FTFSN 211 STIAG 643 RGRGAPQ 709 KSSQSLL 1036 LGS 1237 CMQS 1296 3S 2 AWM SGGR PYYYYGM HSDGKTY NRA LQSPL - S TYYS DVW LY S TF B0 5 00 Fzd FSFST 184 SRING 624 CARAIVGA 743 KSSQSLL 1036 LGS 1237 CMQN 1293 3S 2 YTMS DGSS TGLNRFKA HSDGKTY NRA THWP - TRYA FDIW LY S LTR F0 5 00 Fzd STFTN 363 SAIGT 595 CARDRVTL 796 RSSRSLL 1145 LAS 1231 CIQNT 1270 3S 2 AWM GGGT RGGYSYGT HSNGNT RRA HWPL - S YYA DAFDIW YLR S TR G 05 00 Fzd FTLST 257 SRINY 626 CARDRDIV 786 KSSQSLL 1036 MG 1239 CMQG 1287 3S 2 YNM DGSA VVPAQRG HSDGKTY SYR THWP - N TTYA EGGFDPW LY AS LTF H0 5 00 Fzd FTFSS 228 SAISG 600 CAKGGRD KSSQSLL 1036 LGS 1237 CMQN 1292 3S 2 YAMS SGGS GYKGYFDY 714 HSDGKTY NRA THWP - TYYA W LY S LTL A0 7 00 Fzd FSFRS 178 SAIGT 595 CTTTTVTTS 1026 RTSQSVS 1150 DAS 1187 CQQY 1412 3S 2 YSMS GGGT W SNLA NRA GSSPY - YYA S NF C0 7 00 Fzd FSFSS 183 SHISS 619 CARDGGY 768 RASQSIS 1098 AAS 1175 CQQS 1372 3S 2 YGMS GGAT W SYLN SLQ YSTPL - IDYA S TF F0 7 00 Fzd FTFSS 239 SYISG 658 CARDNGYC 783 RASQAIS 1060 KAS 1228 CQQA 1313 3S 2 YWM DSGY SGGSCYAT SYLA TLD DTFPF - H TNYA YYGMDVR T TF G 07 00 Fzd FTFSS 240 AVISY 476 CARSYYDS 927 KSSQSLL 1036 LGS 1237 CMQT 1299 3S 2 ZZMH DGSN SGYPRKDA HSDGKTY NRA LKAPL - RZYA FDIW LY S TF B0 8 00 Fzd ZSVSS 401 SRINS 625 CARARLLG 750 RSSQYLS 1141 GAS 1213 CQQY 1411 3S 2 NYMS DGSTI GYYTPDR SAYLA SRA GSSPT - SYA MDVW T F F0 8 00 Fzd FTFN 197 ALISS 451 CARDLMV 777 QASQGIS 1049 AAS 1175 CQQS 1367 3S 2 RHAL NGDH GRNKLDY NNLN SLQ YSTPA - S KYYT W S FTF H0 8 00 Fzd FTFSS 225 SGISG 611 CARGRVW 859 RSSQSLL 1135 LGS 1237 CMQG 1287 3S 2 SNM SGSST SSRDYW HSDGYTY NRA THWP - N YYA LY S LTF A0 9 00 Fzd FNIRR 174 SAIGT 595 CARGDSGS 823 RSSESLL 1130 LGS 1237 CTQTV 1433 3S 2 ZNMZ GGGT YRDYW HSDGKTY NRA QFPIT - YYA LY S F B0 9 00 Fzd FTFSS 224 SGISG 612 CARRLIAV 912 RASQGIS 1072 SAS 1252 CQQS 1378 3S 2 SAMH SGTTT AGAEFDP NNLN NLQ YSTP - YYR W S WTF C0 9 00 Fzd FTFSN 214 KAYG 536 CARQYYFD 909 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 SDM GTTE YW SYLN SLQ YSTPL - N YA S TF F0 9 00 Fzd FTFSS 221 SVISS 651 CATASGDF 968 RASQSIG 1086 RAS 1248 CQQT 1398 3S 4 FGMH GGSP DYW SNLN TLES YTTPR - YYA F H0 9 00 Fzd FTFD 191 AIVSY 448 CARQTRG 907 RASQGIS 1072 YAS 1262 CQQS 1345 3S 4 DYA DGTY GTTDGW NNLN SLQ HSPPG - MH KYYS S TF A1 0 00 Fzd FTFSS 222 SAISA 596 CARPIVGA 900 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 HSTH SGDS TAFDIW SYLN SLQ YSTPL - TFYA S TF B1 0 00 Fzd FTZSS 264 SYSSG 674 CARGVVGS 868 RASQSIV 1104 DAS 1186 CQQG 1329 3S 4 YSMN NSGY GAFDIW SYLN NLQ YSAP - TNYA S WTF G 10 00 Fzd FTFSD 208 SAIDG 591 CARAIPGD 742 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YYMS AGRT YDYW SYLN SLQ YSTPL - YYT S TF B1 1 00 Fzd FTFTS 252 GGIIPI 496 CARTGRGY 928 RASQSIG 1085 AAS 1179 CQQS 1373 3S 4 YAM FGIAN YGMDVW SNLD TLQ YSTPR - H YA S TF C1 1 00 Fzd FTFSS 234 SYISG 658 CARAGVAT 741 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 YSMS DSGY IAFDYW SYLN SLQ YSTPL - TNYA S TF D1 1 00 Fzd FTFD 192 SAISG 600 CTTPNYYD 1023 RASQSIS 1098 AAS 1175 CQQS 1372 3S 4 DYG SGGS SR SYLN SLQ YSTPL - MH TYYA S TF F1 1 00 Fzd GTFSS 295 GWIN 558 CARHYYGS 880 ZPZQTZZ 1164 PAS 1242 CQQS 1372 3S 4 YAIS AGNG GSYPDW SHLN SLQ YSTPL - NTTY S TF E1 A 2 00 Fzd FTFST 244 SYISS 671 CARGGLDG 830 RASQGIS 1072 AAS 1179 CQQG 1326 4S 4 YGM SSSAI PIDYR NNLN TLQ NNFPF - H YYA S TF A0 1 00 Fzd FTVSS 260 SLVSF 621 CARLGSTP 887 RASQGIS 1072 AAS 1175 CQQY 1428 4S 4 HSMG DGSK DYW NNLN SLQ YTYPY - EHYA S TF G 01 00 Fzd FTFSS 230 AVISY 475 CASDPVTA 956 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YGM DGSN ATR SYLN SLQ YSTPL - H KYYA S TF C0 2 00 Fzd FSFSS 183 SGISG 610 CAKDGYW 699 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YGMS SGRS SYLN SLQ YSTPL - TYYA S TF D0 2 00 Fzd FTFSS 227 WNG 606 CARPAGSA 899 RASQGIS 1072 DAS 1185 CHQSY 1269 4S 4 YAM GSTG QNWFDP NNLN NLE SIPRTF - H YA W T A0 3 00 Fzd FSFSR 180 SGVG 617 CARDGSW 771 RASQDV 1066 DAS 1191 CQQG 1328 4S 4 YGMS GSGG DTWLA TLET YNIP - STZYA WTF B0 3 00 Fzd YTFTS 388 GIINP 513 CARQIGWE 906 QASQDIS 1047 AAS 1179 CQQAI 1315 4S 4 YAIS SGGS LMPDIW SYLN TLQ SFPLTF - TSYA S C0 3 00 Fzd ZZZTD 403 GGM 510 CANGSYA 732 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YYZQ NZNR QHLW SYLN SLQ YSTPL - GNTG S TF C0 YA 5 00 Fzd FTFSS 239 STISP 649 CAKDKVPY 703 RASQSIS 1098 AAS 1175 CQQS 1372 4S 5 YWM SGLYI SYGPNFDY SYLN SLQ YSTPL - H YQA W S TF G 05 00 Fzd FFFSG 169 QDGS 459 CARVFPLH 934 QASQDIS 1046 KAS 1226 CQQA 1319 4S 5 YWM EKYY DYW NYLN SLES NSFPY - S V TF E0 6 00 Fzd FPFST 176 AGIS 446 CARSGPAA 918 RSSQNVS 1132 GAS 1217 CQHR 1305 4S 5 FSMN WNS MVYYYYG SYLA TRA ANWP - GTIDY MDVW T QTF C0 A 6 00 Fzd FTLSS 256 SAIGT 595 CAAPDYW 686 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 HHM GGGT SYLN SLQ YSTPL - N YYA S TF E0 7 00 Fzd FSFSK 179 SSIDG 629 CARPYYYD 904 QASQDIT 1048 KAS 1229 CQQS 1356 4S 6 KYMT NGDH SSGYDPM NYLN TLES YSAPY - VFYA GDYW TF F0 7 00 Fzd FTVSS 261 SAIGT 595 CAQGTYW 735 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 NYM GGGT SYLN SLQ YSTPL - N YYA S TF C0 8 00 Fzd FTFD 193 SAVS 604 CARGGNY 833 RASQSIS 1102 EAS 1198 CQQT 1397 4S 6 DYYM GNGG GSGDYW ZWLA TLQ YTPPF - N GTFY S TF F0 A 8 00 Fzd GTLN 308 GRIIPI 526 CARDRRGY 795 RASQAIS 1058 DAS 1185 CQQA 1324 4S 6 NHTL FGTA GMDVW NSLA NLE YSFP - S NYA T WTF G 09 00 Fzd FTFSD 208 SGIN 607 CARIGAGG 881 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YYMS WNS AFDIW SYLN SLQ YSTPL - AKIGY S TF B1 V 0 00 Fzd FIFSD 170 AVITS 477 CARNGIAA 896 RASQSIS 1101 GAS 1210 CQQS 1362 4S 6 YYMS GGTF AEDYW TYLS SLES YSPPF - KYYA TF H1 0 00 Fzd FTFSS 226 SGIS 615 CARYSSGG 955 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 SWM WNS SLDYW SYLN SLQ YSTPL - H GSIGY S TF B1 A 1 00 Fzd YZFZZ 400 GRIN 537 CARARSSG 751 RASQSVS 1120 AAS 1180 CQQS 1376 4S 6 ZYMH PNSG WTDAFDI SWLA TLQ YSTPT - GTNY W T F D1 A 1 00 Fzd FTFSS 228 SSISG 632 CARPYSSS 903 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YAMS GGRH RQGDYW SYLN SLQ YSTPL - TYYA S TF F1 1 00 Fzd YIFTD 368 GWIN 564 CARDRPGF 790 RASQSVS 1121 GAS 1213 CQQY 1400 4S 6 YYMH PNSG DPW SYLA SRA AISYTF - GTNY T G A 11 00 Fzd FTFSS 238 SYISG 658 CAKGIRWF 716 RASQSIS 1098 AAS 1175 CQQS 1372 4S 6 YWIH DSGY DPW SYLN SLQ YSTPL - TNYA S TF B1 GWM 2 00 Fzd YIFTD 368 NPNS 583 CASSHYAP 962 RASQGIS 1076 RTS 1250 CQQS 1378 4S 6 YYMH GNTG GMDVW SYLA TLES YSTP - YA WTF C1 2 00 Fzd FTVG 258 SSITT 640 CARGKEGR 844 RASQSIS 1098 AAS 1180 CQQS 1358 4S 7 NNY TSTLY YSNYEAA SYLN TLQ YSIPFT - MS A W T F F1 2 00 Fzd FTFRS 202 SLISG 620 CARREPLY 910 RASQSIS 1098 AAS 1175 CQQS 1372 5S 7 YGM SGDN SSRRGAFDI SYLN SLQ YSTPL - H TNYA W S TF B0 1 00 Fzd FTFSS 234 SAISG 600 CTRTIVGAT 1017 RASQGIS 1072 KAS 1227 CQQS 1360 5S 7 YSMS SGGS PHYW NNLN SLQ YSLPY - TYYA S TF C0 1 00 Fzd FTVSS 262 SAISG 598 CAKGAGY 711 RASQSVS 1119 GAS 1213 CQQR 1338 5S 7 NYMS SGAT GSGSWQA SSYLS SRA YKSYT - TTYA AW T F F0 1 00 Fzd YSFTN 370 GRIIPI 527 CARGTFLE 866 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAM FGTA WLLTNYG SYLN SLQ YSTPL - H ZYA MDVW S TF B0 2 00 Fzd GTFSS 297 GWIG 554 CATGWPR 971 RASQSVS 1116 NTS 1241 CQHY 1308 5S 8 YVIS PHNG YYYGMDV SNLA NRA NNWP - NTNY W T FTF D0 A 2 00 Fzd YTFTS 392 GGIIPI 500 CARLPYYD 889 RASQSVS 1122 DAS 1188 CQQR 1336 5S 8 YYMH FGTA FWSGYYG TNLA NRA SNWP - ZYA GRTGFDY T PQITF G W 02 00 Fzd YTFTY 394 GWIN 558 CARASLYY 753 QASQDIS 1044 AAS 1175 CQQS 1372 5S 8 RYLH AGNG DYVWGSY HYLN SLQ YSTPL - NTTY RHYYFDY S TF H0 A W 2 00 Fzd GTFSS 295 GIINP 512 CATSFGGG 975 RASQSIN 1091 GAS 1213 CQQY 1413 5S 8 YAIS SGGR WIVVDTSL SNLA SRA GSSPY - TTYA WYW T TF B0 3 00 Fzd GSFS 275 GGIIPI 499 CRVDAFDI 1009 RASQSVS 1119 DTS 1194 CQQY 1408 5S 8 GYAIS FGTA W SSYLS NRA GSSPI - NYA T TF C0 3 00 Fzd FTFTS 251 GGIIPI 499 CARSSGW 923 RTSQSISS 1149 AAS 1181 CQQSF 1343 5S 8 SAVQ FGTA QNRFAFDI YLN TSQ SSWTF - NYA W S E0 3 00 Fzd YTFTY 394 GWIN 557 CATDLPVR 970 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 RYLH AGNG KGFTYYDIL SYLN SLQ YSTPL - NTKY TGSYGMD S TF F0 S VW 3 00 Fzd YTFTN 383 GGIIPI 498 CARGLRYF 848 QASHDI 1039 AAS 1175 CQQS 1365 5S 8 NFMH FGTA DWPQGIY NIALN SLQ YSSPL - NHA YYYGMDV S TF B0 W 4 00 Fzd YTFTS 392 GRIN 537 CARGGLLF 831 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YYMH PNSG DYW SYLN SLQ YSTPL - GTNY S TF C0 A 4 00 Fzd FTFST 246 STIGT 645 CARVGWL 938 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YSMS GGGT RFLDYW SYLN SLQ YSTPL - YYA S TF D0 4 00 Fzd GTFSS 295 GWM 585 CARNNFLR 897 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS SPSSG AFDIW SYLN SLQ YSTPL - NAGY S TF G A 04 00 Fzd FAFSS 167 SRIDT 622 CARAPSYS 747 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAMS DGST SGWYVRW SYLN SLQ YSTPL - TYVA S TF A0 5 00 Fzd YTFTY 395 GIINP 513 CARELLPM 812 RASQGIS 1072 RAS 1247 CQQA 1320 5S 8 YAM SGGS TTVTSPFI NNLN SLQ NSYPL - H TSYA W S TF C0 5 00 Fzd GTFSS 295 GGIIPI 499 CAIRAFDI 694 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 YAIS FGTA W SYLN SLQ YSTPL - NYA S TF E0 5 00 Fzd ZTFSZ 402 SSISS 635 CARVRSKA 947 RASQSVS 1119 AAS 1172 CQQY 1424 5S 8 YDM SSHY VAGTLPKR SSYLS RRA SNWP - H KYYA LFDIW T FTF C0 6 00 Fzd YTFTS 392 GWM 583 CARGNPTS 850 QASQDIS 1045 SAS 1253 CQQS 1354 5S 8 YYMH NPNS GHIVVVPA NRLN RLQI YRTPR - GNTG ATFSDYW TF E0 YA 6 00 Fzd GTFSZ 302 GWM 579 CARWAFPI 950 RASQSIS 1098 AAS 1175 CQQS 1372 5S 8 ZTIS NPDS PNAFDIW SYLN SLQ YSTPL - GKTG S TF G YA 06 00 Fzd YTFTN 383 GGIFP 494 CARDRPSS 791 RASQGIS 1072 DAS 1193 CQQSF 1342 5S 8 NFMH IYGIS SWYAFDY NNLN TLQ SAPITF - TYA W T H0 6 00 Fzd GTFSZ 301 GGIIPI 499 CARGGLLR 832 RASQSIS 1097 GAS 1217 CQQY 1407 5S 9 YAIS FGTA FGDGWG SKSLA TRA GIAPT - NYA MGMDVW T F A0 8 00 Fzd YTFTD 375 GWIN 558 CARASSWY 755 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YHM AGNG LHYYYGM SYLN SLQ YSTPL - H NTTY DVW S TF C0 A 8 00 Fzd FIFSZ 171 SSISA 631 CARRGYSS 911 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YAMS AGAY GWRDAFD SYLN SLQ YSTPL - KYYA IW S TF E0 8 00 Fzd YTFTS 392 GWIN 558 CAKDVNY 710 QASQGIS 1050 AAS 1175 CQQT 1395 5S 9 YYMH AGNG W NYLN SLQ YSTPT - NTTY S TF F0 A 8 00 Fzd GTFSS 295 GRIIPI 531 CARDRLAF 789 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YAIS LGTP DYW SYLN SLQ YSTPL - NYA S H0 TF 8 00 Fzd FAFSS 166 SAISV 602 CARWGKR 952 RASQSIG 1085 RAS 1249 CQQS 1375 5S 9 HWM SGGT LRGSPYYF SNLD TLQ YSTPS - H TFYA DYW S F A0 9 00 Fzd FTFSI 209 SGIS 614 CARGPLPT 852 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YGM WNS KIGGHYM SYLN SLQ YSTPL - H GNIG DVW S TF F0 YA 9 00 Fzd FTFST 241 AVMY 478 CARLSYYY 891 RASQGIS 1072 AAS 1175 CQQG 1327 5S 9 XWM SGGT DSSGPKGD NNLN SLQ NNFPL - S TYYA AFDIW S TF B1 0 00 Fzd FSLSS 185 SSISS 636 CARSGMV 917 RASQDIG 1061 AAS 1175 CQKY 1310 5S 9 YGM SSSYI KWLRSFDY SFLA SLQ NRAPF - H YYA W S TF C1 0 00 Fzd FTFTS 249 GVIN 552 CARGYGDY 873 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 SAM PGSG VWGENYF SYLN SLQ YSTPL - Q GTSY DYW S TF E1 N 0 00 Fzd YTLSN 397 GWIS 568 CARFDYFG 816 RASQSIS 1098 AAS 1175 CQQS 1372 5S 9 YGIS AYNG GMDVW SYLN SLQ YSTPL - DTKY S TF F1 A 0 00 Fzd YTFTR 385 GGIIP 495 CAADRSPY 679 RASQGIS 1072 QAS 1244 CQQS 1352 5S 9 YAVH FFNT YYDSSGYY NNLN SLD YNVPY - VNYA PDAFDIW S TF G 10 00 Fzd FTFSS 229 SGIS 615 CAKGSLLL 720 RASQSIS 1092 DAS 1192 CQQS 1350 5S 9 YDM WNS GYYGMDV NNLN TLK YNTPR - N GYIGY R TF A1 A W 1 00 Fzd FTZSS 263 SSISG 633 CAREAGTT 805 RASQSIS 1098 AAS 1175 CQQS 1372 5S 10 YDM LGGS GGWFDP SYLN SLQ YSTPL - H TYZA W S TF B1 2 00 Fzd FTFSD 204 STIGP 644 CARASTSG 756 RASQSVS 1123 GAS 1217 CQQY 1406 5S 10 HYM AGDT DYSLW TSYLA TRA GASP - D YYP T WTF D1 2 00 Fzd YTFTN 384 GLVC 519 CARRTSAS 915 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YCTR PSDG DIW SYLN SLQ YSTPL - STSYA S TF B0 1 00 Fzd FTFTZ 253 GGFD 492 CTTDPLELP 1020 RASQGIS 1076 SAS 1252 CQQAI 1315 6S 10 SAVQ PEDG WYW SALA NLQ SFPLTF - ETIYA S C0 1 00 Fzd YTFTG 379 GIINP 514 CARDLTYY 781 RASQSVT 1124 GAS 1217 CQQY 1416 6S 10 YYMH SSGR YDSSGHSP SSLA TRA NDWP - TDYA LGAFDIW T PTF E0 1 00 Fzd FTFSD 203 AGISG 443 CARDSDF 797 KSSQSVL 1037 STN 1257 CQHR 1306 6S 10 FGM GGGS WYYYGMD YSSNNK TRS NFF - N TDYA VW NYLA S G 01 00 Fzd VSFS 366 AYINS 482 CAREEWEL 807 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 GYA GSSE FGMDVW SYLN SLQ YSTPL - MH MNY S TF B0 A 2 00 Fzd YTVTS 399 GGIIPI 497 CAKGGQW 713 RASQSIS 1098 AAS 1175 CQQS 1372 6S 10 YAM FGTA LYGMDVW SYLN SLQ YSTPL - H KYA S TF G 02 01 Fzd YTFTS 392 GWVS 588 CARDEGA 766 RASQSIS 1098 AAS 1175 CQQS 1372 4S 1 YYMH PSSG GYYYYYM SYLN SLQ YSTPL - NTAY DVW S TF A0 A 1 01 Fzd FTFSN 216 SAIGT 595 CATAYRRP 969 RSSQSLL 1134 LGS 1237 CMQN 1293 4S 2 YAMT GGGT GGLDVW HSDGKTY NRA THWP - YYA LY S LTR C0 1 01 Fzd FTFSS 227 SVIST 652 CARGGSSD 838 RASQSIS 1098 AAS 1175 CQQS 1372 4S 4 YAM SGDT VR SYLN SLQ YSTPL - H VLYT S TF F0 1 01 Fzd FTFSN 217 SYISS 672 CARAALGY 737 RASQGIS 1072 AAS 1171 CQQS 1363 4S 4 YGM SSSTI CTGGVCPP NNLN RLQ YSPPL - H YYA VDYW S TF H0 1 01 Fzd YTFTN 383 GIIZP 517 CAKGDYG 712 RASQSIS 1098 AAS 1175 CQQS 1372 4S 7 NFMH GGGR ALDYW SYLN SLQ YSTPL - TIYA S TF C0 3 01 Fzd FNFSS 173 SVIYG 653 CARGGSG 836 RASQSIS 1098 AAS 1175 CQQS 1372 4S 7 YTMR GGNT GNLSYW SYLN SLQ YSTPL - NYA S TF D0 3 01 Fzd YTFTN 383 GGIIP 506 CARLVVRG 893 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 NFMH LFGTA GYGMDV SYLN SLQ YSTPL - NYA W S TF F0 3 01 Fzd GTFSS 295 GWIS 575 CARADDYY 738 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YAIS SFNG DSSGYYYG SYLN SLQ YSTPL - NTKY FDYW S TF A0 A 4 01 Fzd FTFSS 235 SRING 623 CARGWAG 871 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 YTMN DGSN FDYW SYLN SLQ YSTPL - TNYA S TF C0 4 01 Fzd HTFS 312 GWIN 558 CARDLSPM 779 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 GYHI AGNG VRGVISGM SYLN SLQ YSTPL - H NTTY DVW S TF D0 A 4 01 Fzd YTFTN 383 GIISP 515 CAKGDYG 712 RASQSIS 1098 AAS 1175 CQQS 1372 4S 8 NFMH GGGR ALDYW SYLN SLQ YSTPL - TIYA S TF E0 5 01 Fzd FTFG 195 SSLS 641 CARDSSSG 798 RASQSIS 1095 AAS 1175 CLQH 1275 4S  10 NYD WNS WYASYYG RYLN SLQ HSYPF - MN GTIVY MDVW S TF E0 A 6 02 Fzd YTLTT 398 GWM 582 CARGALG 819 QASQDIS 1046 AAS 1173 CQESY 1302 7S 5 WYM NPNS MDVW NYLN SLH SSPYT - X GNTA T F C0 YA 2 02 Fzd YTFTG 378 GWM 583 CARGTGGF 867 RASQSIS 1098 AAS 1175 CQQS 1379 7S 8 HYM NPNS DYW SYLN SLQ YSTPY - H GNTG S TF E0 TA 3 02 Fzd YTFTG 377 GWM 580 CARSTPFD 925 RASHDIG 1057 AAS 1179 CQQS 1355 7S 8 HYIH NPISG PW TFLA TLQ YRTPY - NTGY S TF F0 A 3 02 Fzd YTFTH 380 GWIN 559 CARGDYDF 824 QATQNI 1051 KAS 1229 CQQS 1372 7S 8 SYIH AKSG WSGYHEY KKYLN TLES YSTPL - GTFY YYYGMDV TF G A W 03 02 Fzd YTFTS 392 GWIN 564 CARAPLDG 746 RSSQSLL 1138 LGS 1237 CMQA 1282 7S 8 YYMH PNSG SGSYYVD HSNGYN NRA LQTPQ - GTNY W YLD S TF H0 A 3 02 Fzd YTFTN 382 GWIS 571 CARDCSGG 765 RASQSIS 1093 AAS 1169 CQQA 1323 7S 8 HFMH PNRG SCYSHFDY RSLA NLQ YSFPQ - GTNY W S TF A0 A 4 02 Fzd FTVG 259 SAIGT 595 CAKDITPY 702 RASQAIS 1059 AAS 1175 CQQTF 1383 7S 8 SWY GGGT GDYSILSH NYLN SLQ SPPLT - MS YYA W 5 F B0 4 02 Fzd YTFTS 386 GGIIPI 503 CARDSSSW 799 RASQGIN 1067 QAS 1243 CQQT 1393 7S 8 HWM FGTT YSYYYYYM NYLA NLE YSSPL - H NYA DVW S TF C0 4 02 Fzd YTFTT 393 GWIY 578 CTTDLRYD 1019 QASQDI 1040 AAS 1175 CQQS 1377 7S 8 YFMH PNSG SSGPAAFDI DNYLN SLQ YSTPV - GTKY W S TF D0 A 4 02 Fzd FTFSD 205 SGISG 609 CATYGDFG 978 RASQGIR 1069 AAS 1179 CQQA 1324 7S 8 HYMS SGGT YFDLW NDLG TLQ YSFP - TYYA S WTF E0 4 02 Fzd GSFST 279 GRIIP 532 CVKDRAW 1027 RASQGIR 1068 AAS 1174 CQQS 1359 7S 8 SVFG LFGTT GFDYW NDLA SLQ YSKPT - NYA R F C0 5 02 Fzd YTFTS 392 GWIN 560 CARGGFVF 828 QASQDIS 1046 ASS 1182 CQQS 1356 7S 8 YYMH PKSG DYW NYLN TLQ YSAPY - GTNY T TF D0 A 5 02 Fzd GTFSS 295 GMIN 523 CARQAGLH 905 RASQGIT 1077 AAS 1168 CQQY 1422 7S 8 YAIS PSGG CSSTSCYLG KSLA NLQ NTFPI - STTYA NWFDPW L TF E0 5 02 Fzd GTFN 290 GGIIP 508 CAKGNWA 719 RASQSIS 1100 GAS 1217 CQQY 1411 7S 8 RYGIS RLGA FDIW TYLA TRA GSSPT - TDYA T F F0 5 02 Fzd GTFSS 295 GWIS 572 CARGVWT 869 RASQSIS 1098 YAS 1261 CQQS 1368 7S 8 YAIS PYNG TPMGGGG SYLN SLQ YSTPF - NTKY NWFDPW N TF G A 05 02 Fzd GTFG 286 GWIN 564 CARETTDY 814 RASQSIG 1088 DAS 1185 CQQA 1317 7S 8 NYGI PNSG YYGMDVW TYLN NLE NSFPL - N GTNY T TF H0 A 5 02 Fzd GTFSS 294 GVID 550 CARVLPGD 941 RASQGIS 1072 KAS 1226 CQQA 1316 7S 8 YAIN PSTG SSGWYRG NNLN SLES NSFPI - GTNY YYYYYGM TF A0 A DVW 6 02 Fzd GTFTS 306 GWIN 566 CARDLDSG 776 RASQGV 1078 DAS 1186 CQQH 1330 7S 8 YPIS TYNG FDLW GDYLA NLQ NAYPL - NTIYA S TF C0 6 02 Fzd GTFSS 295 GWIS 569 CARGGYSY 842 RASQDIS 1064 KAS 1229 CQQS 1348 7S 8 YAIS AYNG GTVFDYW SWLA TLES YGAPL - HTNY TF D0 A 6 02 Fzd YTFTK 381 GGIIPI 499 CARGLPPA 847 RASQNV 1082 SAS 1252 CQQS 1368 7S 8 DYM FGTA AGGGGYF NDWLA NLQ YSTPF - H NYA QHW S TF E0 6 02 Fzd FTFSS 227 AVTW 480 CAKDLVPY 705 RASQSIS 1098 GAS 1207 CQQS 1372 7S 8 YAM YDGS CSGGSCPP SYLN NLQ YSTPL - H NKYY SGW S TF F0 A 6 02 Fzd YTFTD 376 GWM 584 CARGKSGS 846 RASQSV 1111 GTS 1222 CQQY 1404 7S 5 YYMH SPNS FDYW NNTYVA TRA DTSPP - FA GNAG T TF G 06 02 Fzd YTFTG 377 GIINP 513 CARGFCSG 825 RASQSIS 1098 AAS 1175 CQQS 1382 7S 5 HYIH SGGS GSCLWYG SYLN SLQ YTTPF - TSYA MDVW S TF H0 6 02 Fzd GTFG 287 GGIIPI 499 CAKDNGW 706 RASQSIS 1099 AAS 1175 CQQS 1379 7S 5 SYAIT FGTA YFDLW TNVN SLQ YSTPY - NYA S TF A0 7 02 Fzd GTFSS 295 GRIN 1461 CARATRVS 1472 KSSQSVL 1498 STN 1257 CQQY 1544 9S 1 YAIS PHNG AAGTVHF HSSNNK TRS YSTPF - NTNY QHW NYLA S TF B0 A 1 02 Fzd YTFTR 1452 GWM 583 CARVRFLE 1494 RASQSLS 1512 DAS 1520 CQQAI 1315 9S 2 YYIH NPNS EMDVW SWLA TLQ SFPLTF - GNTG S D0 YA 1 02 Fzd GTFSS 1457 GIINP 513 CARGDIVA 1478 RASQDIS 1500 GAS 1522 CQQA 1318 9S 2 YGIS SGGS TMGMKKV NNLN HLQ NSFPV - TSYA DYYYYMD T TF C0 VW 2 02 Fzd YTFTR 1453 GWM 583 CARGIGYW 1481 RASQGIS 1074 AAS 1515 CLQYN 1528 9S 2 YYLH NPNS NYLA RLQ TYPW - GNTG T TF F0 YA 2 02 Fzd GTFST 298 GDIIPI 1458 CARELGLG 1476 RSSQSLL 1138 LGS 1525 CMQA 1281 9S 2 YAIS FGSA WFDPW HSNGYN SRA LQTPL - NYA YLD S TF H0 2 03 Fzd YTFTD 1449 GWM 1467 CARGDINY 1477 RASQSIS 1098 KAS 1523 CQQAI 1315 0S 7 YYMH NPNS GNFDYW SYLN TLH SFPLTF - GSTG N A0 YA 2 03 Fzd YTFTD 1449 GWM 583 CARQGGSY 1488 RASQSIT 1511 KTS 1524 CQQG 1531 0S 3 YYMH NPNS SMGLDPW TYLN SLQ DSFPY - GNTG S TF B0 YA 2 02 Fzd YTFTG 379 GWIN 1463 CARSYYGV 1492 RASQSIS 1098 AAS 1516 CQQSF 1532 9S 3 YYMH PNSG IDAFDIW SYLN SLQ RLPLT - NTGY T F E0 A 3 02 Fzd YTFTN 1451 GWM 583 CAREDDF 1474 RASQSIIS 1509 AAS 1175 CQQS 1535 9S 3 YYMH NPNS WSGGGM YLN SLQ WRFP - GNTG DVW S YTF G YA 03 03 Fzd FTFSD 1438 SAISG 1468 CAREGLRG 1475 KSSQSVL 1037 WA 1526 CQQY 1545 0S 3 YYMS SGHS WSIFDIW YSSNNK STR YSTPP - TYYA NYLA ES TF E0 3 02 Fzd YTFTD 1447 GWA 1462 CARSRLRW 1491 RASQTIS 1513 DAS 1185 CQQS 1536 9S 3 HYFH NPSS DWYFDLW SYLN NLE YSIPLT - GNTG T F D0 TA 5 03 Fzd FSFSS 1437 SAIGT 595 CANPKHY 1470 RASQGV 1505 AAS 1175 CQQY 1543 0S 3 HAMS GGGT W STYLA SLQ YSSPQ - YYA S TF H0 3 02 Fzd YTFSR 1446 GWM 583 CARGGHT 1479 RASQSVS 1120 AAS 1175 CQQA 1529 9S 3 HYIH NPNS NHW GYSSGWY SLQ FRFPP - GNTG SWLA S TF B0 YA 6 02 Fzd YWFT 1455 GWM 1465 CARRSSSW 1489 RASQNIN 1506 AAS 1175 CQQY 1542 9S 3 ASYM KPDS GWYFDLW SWLA SLQ YSFPL - H GNTG S TF E0 YA 6 2 Fzd YTFAK 1444 GWM 583 CARHKRHT 1487 RASQSIS 1098 AAS 1175 CQQS 1533 9S 3 YYIH NPNS PYAFDIW SYLN SLQ HSTPL - GNTG S TF H0 YA 6 02 Fzd YTFTD 1448 GWIS 570 CARGSGYF 1486 RASQSIS 1510 GAS 1215 CQQA 1324 9S 3 SYIH AYNG DLW KWLA TLQ YSFP - NTNY S WTF G A 07 02 Fzd YTFTG 378 GWM 583 CARVGDY 1493 RASRTVY 1514 DAS 1519 CQQS 1537 9S 3 HYM NPNS DRFNWYF NFLA NLR YSTPP - H GNTG DLW T TF H0 YA 8 02 Fzd GTFSS 1456 GWIS 570 CARANRGL 1471 RASQSIA 1507 GAS 1211 CQQS 1351 9S 3 YAIT AYNG RKNYYYG RYLN SLQ YNTP - NTNY MDVW S WTF F0 A 9 03 Fzd YTFTS 1454 GIINP 1459 CARWTTV 1495 RASQGIR 1502 DAS 1518 CQQSS 1534 0S 3 SYIH SGGG VTGAAFDI NDLN NLG RIPPTF - AVYA W T F0 4 02 Fzd YSFTG 1442 GWIN 564 CARDHGT 1473 RASQGIS 1503 AAS 1175 CQQS 1538 9S 3 YYLH PNSG MIAVAGTF KYLA SLQ YSTP - GTNY DYYYYMD S WTF A1 A VW 0 02 Fzd YTFN 1445 GIVN 1460 CARGGNY 1480 QASQDIS 1046 GAS 1521 CQQT 1540 9S 3 GYYM PSGG GRWLQPW NYLN ALR KSFPL - H GTNY YFDLW S TF B1 A 1 02 Fzd HTFTS 1440 GWM 1466 CARGLGYF 1483 RASQDIS 1501 AAS 1517 CQQA 1324 9S 3 HYM NPNS DLW RGLG TLY YSFP - H ANAG R WTF D1 YA 1 03 Fzd YSFTN 1443 GWM 583 CARSPDF 1490 RASQSIG 1508 AAS 1175 CQQA 1530 0S 7 YYMH NPNS WSGEGYF NYLN SLQ NSFPL - GNTG DLW S TF H0 YA 5 03 Fzd YMFT 1441 GRIIPI 529 CARGIHGD 1482 RASQAIG 1499 AAS 1175 CQQY 1541 0S 7 GHD LGIAN YGLDYYYM RRLA SLQ DTYW - MH YA DVW S TF A0 6 02 Fzd YTFTG 379 GWM 583 CARGMEY 1484 RASQGIS 1076 AAS 1179 CLQYN 1528 9S 7 YYMH NPNS W SYLA TLQ TYPW - GNTG S TF C1 YA 2 03 Fzd YTFTG 1450 GWM 1464 CARGPADF 1485 RASQGIS 1504 DAS 1190 CQQS 1539 0S 7 YYIH DPNS WSGYKND SWLA SLQ YSTPY - GYTG YFDFW S SF C0 YA 6 4A12 Fzd GYTFT 1439 WIYP 1469 CVRSAWG 1496 KASQDV 1497 WA 1527 QQYS 1546 5 NYDI RDGS FAY GTAVA STR TYPLT N TKYN HT EKFK G

TABLE 1B Anti-Fzd Antibody Clone IDs, Heavy Chain (HC) and Light Chain (LC) Seq ID Nos, and Binding Characteristics Clone ID HC SID NO LC SID NO Confirmed Binding 001S-B01 1 38 Fzd1, 2, 7, 9 001S-E02 2 39 Fzd1, 2, 7 001S-G02 3 40 Fzd1, 2, 7 001S-H02 4 41 Fzd1, 2, 7 001S-A03 5 42 Fzd1, 2, 7, 9 001S-B03 6 43 Fzd1, 2, 7 004S-G06 7 44 Fzd5, 8 002S-B01 8 Fzd1 002S-C02 9 Fzd1 002S-E02 10 Fzd1 002S-G02 11 Fzd1 002S-F03 12 Fzd1 002S-A04 13 Fzd1 002S-B04 14 Fzd1 002S-D04 15 Fzd1 004S-H04 16 45 Fzd5 001S-A04 17 46 Fzd1, 2, 5, 7, 8 003S-E07 18 47 Fzd2 003S-D10 19 48 Fzd4 004S-B08 20 49 Fzd6 004S-D08 21 50 Fzd6 004S-C09 22 51 Fzd6 004S-F10 23 52 Fzd6 004S-A11 24 53 Fzd6 004S-A12 25 54 Fzd6 005S-B07 26 55 Fzd9 005S-D08 27 56 Fzd9 005S-E09 28 57 Fzd9 005S-H10 29 58 Fzd9 005S-B11 30 59 Fzd9 005S-D11 31 60 Fzd9 014S-G02 32 61 Fzd6 014S-B04 33 62 Fzd8 014S-B06 34 63 Fzd9 014S-G06 35 64 Fzd10 014S-A07 36 65 Fzd10 017S-B09 37 Fzd8 004S-D01 129 130 Fzd4 004S-E09 131 132 Fzd6 004S-F09 Fzd6 004S-H09 Fzd6 004S-B10 Fzd6 004S-C10 Fzd6 004S-F10 Fzd6 004S-G10 Fzd6 004S-A11 Fzd6 004S-B11 n.b. 004S-D11 Fzd6 004S-E11 n.b. 004S-F11 Fzd6 004S-G11 Fzd6 004S-A12 Fzd6 004S-B12 Fzd6 004S-C12 n.b. 004S-D12 n.b. 004S-F12 n.b. 004S-F12 n.b. 004S-G12 n.b. 005S-B02 n.b. 005S-C02 n.b. 005S-D02 Fzd5, 8 005S-E02 Fzd5, 8 005S-H02 Fzd5, 8 005S-A03 Fzd5, 8 005S-C03 n.s. 005S-E03 n.s. 005S-F03 Fzd8 005S-B04 Fzd5, 8 005S-F04 n.b. 005S-G04 Fzd5, 8 005S-H04 n.b. 005S-E05 n.b. 005S-G05 Fzd5, 8 005S-H05 Fzd5, 8 005S-D06 Fzd8 005S-F06 n.b. 005S-G06 n.b. 005S-A07 Fzd9, 10 005S-B07 Fzd9 005S-A08 Fzd9 005S-B08 Fzd9 005S-D08 Fzd9 005S-E08 Fzd9 005S-F08 n.b. 005S-C09 Fzd9 005S-D09 Fzd9 005S-E09 Fzd9 005S-F09 Fzd9 005S-A10 Fzd9 005S-B10 Fzd9 005S-E10 Fzd9 005S-H10 Fzd9 005S-B11 Fzd9 005S-D11 Fzd9 005S-G11 n.b. 005S-H11 n.b. 005S-E12 Fzd10 006S-A01 Fzd10 006S-H01 n.b. 006S-A02 Fzd10 006S-D02 n.b. 006S-H02 Fzd10 006S-A03 n.b. 006S-B03 n.b. 006S-C03 n.b. 014S-A01 Fzd1, 2, 7 014S-B02 n.b. 014S-G02 Fzd6 014S-B03 n.b. 014S-C03 Fzd1, 2, 7 014S-A04 n.b. 014S-B04 Fzd8 014S-B05 Fzd5, 8 014S-B06 Fzd9 014S-F06 n.s. 014S-G06 Fzd10 014S-A07 Fzd10 017S-E08 Fzd8 017S-H08 n.b. 017S-A09 n.b. 017S-B09 Fzd8 018S-F06 Fzd4 018S-H06 n.b. 018S-B07 n.b. 017S-A10 n.b. 017S-B10 n.b. 017S-D10 n.b. 018S-H08 n.b. 018S-B09 Fzd5, 8 021S-A01 n.b. 021S-E02 Fzd5, 8 021S-G02 n.s. 021S-A03 n.b. 029S-B01 n.b. 029S-D01 Fzd1, 2, 7 029S-C02 Fzd1, 2, 7 029S-H02 Fzd1 030S-A02 Fzd7 029S-E06 Fzd2, 6, 3 030S-F04 Fzd3 030S-H05 Fzd7 030S-A06 Fzd1, 2, 7, 5 029S-C12 Fzd7 030S-C06 Fzd1 001S-A01 Fzd1, 2, 7 001S-H01 Fzd1, 2, 7

In certain embodiment, the Fzd binding domain may be selected from any binding domain that binds a Fzd with an affinity of, e.g., a K_(D) of at least about 1×10⁻⁴ M, at least about 1×10⁻⁵ M, at least about 1×10⁻⁶ M, at least about 1×10⁻⁷ M, at least about 1×10⁻⁸ M, at least about 1×10⁻⁹ M, or at least about 1×10⁻¹⁰ M. In certain embodiment, the Fzd binding domain may be selected from any binding domain that binds one or more Fzd receptor at high affinity, e.g., a K_(D) of less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, less than about 1×10⁻⁹ M, or less than about 1×10⁻¹⁰ M. In certain embodiment, the Fzd binding domain may be selected from any binding domain that binds Fzd at high affinity, e.g. a K_(D) of less than or equal to about 1×10⁻⁴ M, less than or equal to about 1×10⁻⁵ M, less than or equal to about 1×10⁻⁶ M, less than or equal to about 1×10⁻⁷ M, less than or equal to about 1×10⁻⁸ M, less than or equal to about 1×10⁻⁹ M, or at least about 1×10⁻¹⁰ M in the context of a Wnt surrogate molecule.

Suitable Fzd binding domains include, without limitation, de novo designed Fzd binding proteins, antibody derived binding proteins, e.g. scFv, Fab, etc. and other portions of antibodies that specifically bind to one or more Fzd proteins; VHH or single domain antibody derived binding domains; knottin-based engineered scaffolds; norrin and engineered binding fragments derived therefrom, naturally occurring Fzd binding domains, and the like. A Fzd binding domain may be affinity selected to enhance binding to a desired Fzd protein or plurality of Fzd proteins, e.g. to provide tissue selectivity.

In some embodiments, the Fzd binding domain binds to one, two, three, four, five or more different frizzled proteins, e.g. one or more of human frizzled proteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10. In some embodiments, the Fzd binding domain binds to Fzd1, Fzd2, and Fzd 7. In some embodiments, the Fzd binding domain binds to Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8. In other embodiments the Fzd binding domain is selective for one or more frizzled protein of interest, e.g. having a specificity for the one or more desired frizzled protein of at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more relative to other frizzled proteins.

In certain embodiments, the Fzd binding domain comprises the six CDR regions of the pan specific frizzled antibody OMP-18R5 (vantictumab). In certain embodiments, the Fzd binding domain is an scFv comprising the six CDR regions of the pan-specific frizzled antibody OMP-18R5 (vantictumab). See, for example, U.S. Pat. No. 8,507,442, herein specifically incorporated by reference. For example, the CDR sequences of OMP-18R5 include a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:270), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:677), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:1033), and (ii) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:1152) or SGDNIGSFYVH (SEQ ID NO:1153), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO:1200) or DKSNRPSG (SEQ ID NO:1201), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:1435) or QSYANTLSL (SEQ ID NO:1436). In particular embodiments, the Fzd binding domain is an antibody or derivative thereof, including without limitation scFv, minibodies, VHH or single domain antibodies (sdAb) and various antibody mimetics comprising any of these CDR sequences. In certain embodiments, these CDR sequences comprise one or more amino acid modifications.

In other embodiments, the Fzd binding domain comprises a variable region sequence, or the CDRs thereof, from any of a number of frizzled specific antibodies, which are known in the art and are commercially available, or can be generated de novo. Any of the frizzled polypeptides can be used as an immunogen or in screening assays to develop an antibody. Non-limiting examples of frizzled binding domains include antibodies available from Biolegend, e.g. Clone CH3A4A7 specific for human frizzled 4 (CD344); Clone W3C4E11 specific for human Fzd9 (CD349); antibodies available from Abcam, e.g. ab64636 specific for Fzd7; ab83042 specific for human Fzd4; ab77379 specific for human Fzd7; ab75235 specific for human Fzd8; ab102956 specific for human Fzd9; and the like. Other examples of suitable antibodies are described in, inter alia, US Patent application 20140105917; US Patent application 20130230521; US Patent application 20080267955; US Patent application 20080038272; US Patent application 20030044409; etc., each herein specifically incorporated by reference.

The Fzd binding region of a Wnt surrogate molecule may be an engineered protein that is selected for structural homology to the frizzled binding region of a wnt protein. Such proteins can be identified by screening a structure database for homologies. The initial protein thus identified, for example the microbial Bh1478 protein. The native protein is then engineered to provide amino acid substitutions that increase affinity, and may further be selected by affinity maturation for increased affinity and selectivity in binding to the desired frizzled protein. Non-limiting examples of frizzled binding moieties include the Fz27 and Fz27-B12 proteins.

In particular embodiments, a Wnt surrogate molecule comprises an LRP5/6 binding domain, e.g., an anti-LRP5/6 antibody, or antigen-binding fragment thereof, fused to a polypeptide that specifically binds to one or more Fzd receptors. In particular embodiments, the polypeptide that specifically binds to LRP5/6 is an antibody or antigen-binding fragment thereof. If certain embodiments, it is an antibody or antigen-binding fragment thereof disclosed in the U.S. provisional patent application No. 62/607,879, titled, “Anti-LR5/6 Antibodies and Methods of Use,” filed on Dec. 19, 2017, which is incorporated herein by reference in its entirety. In particular embodiments, the LRP5/6 binding domain comprises the three heavy chain CDRs and/or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to LRP5 and/or LRP6 provided in Table 2A. In particular embodiments, the LRP5/6 binding domain comprises the three heavy chain CDRs and/or the three light chain CDRs disclosed for any of the illustrative antibodies or fragments thereof that bind to one or more Fzd receptor provided in Table 2A, wherein the CDRs collectively comprise one, two, three, four, five, six, seven, or eight amino acid modifications, e.g., substitutions, deletions, or additions. In certain embodiments, the LRP5/6 binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so Table 2A only includes the three heavy chain CDRs. In certain embodiments, the LRP5/6 biding domain comprises the three heavy chain CDRs shown in Table 2A or variants wherein the CDRs collectively comprise one, two, three, four, five, six, seven or eight amino acid modifications. In particular embodiments, the LRP5/6 binding domain comprises the heavy chain fragment and/or light chain fragment of any of the illustrative antibodies or fragments thereof that bind to LRP5 and/or LRP6 provided in Table 2B or SEQ ID NOs:66-88 or 133 (or an antigen-binding fragment or variant of either). In certain embodiments, the LRP5/6 binding domain is an Fab or was derived from an Fab, so Table 2B includes VH and CH1 sequence, but not CH2 or CH3 sequences. In certain embodiments, the LRP5/6 binding domain is a VHH or sdAb or was derived from a VHH or sdAb, so Table 2B includes the VHH domain. In certain embodiments, the LRP5/6 binding region is a polypeptide, e.g., an antibody or antigen-binding fragment thereof, that competes with one of these antibodies for binding to LRP5 and/or LRP6.

TABLE 2A Anti-LRP5/6 Antibody Clone IDs and CDR sequences. Confirmed CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 Clone ID Binding CDRH1 SEQ ID CDRH2 SEQ ID CDRH3 SEQ ID CDRL1 SEQ ID CDRL2 SEQ ID CDRL3 SEQ ID 001S-CO8 LRP6e1e2 YTISNYYIH 1682 GMINPSGGST 1762 CAIVRGKKWYFDLW 1842 RASQYISNYLN 2098 AASSLQS 2110 CQQSYITPLTF 2160 TYA 001S-C10 LRP6e1e2 RTFGTYPNG 1632 AAISWGGRTA 1700 CYARTVIGGFGAFRA 2061 YA HW 001S-D10 LRP6e1e2 RTFSRYAMA 1642 AAIRWSGGGT 1689 CAASMEAMNSLRVN 1836 YYA KERYYQSW 001S-E10 LRP6e1e2 LTFSNAAMA 1614 AAISRSGANTA 1696 CTLVNEIKTWW 2039 YS 001S-F10 LRP6e1e2 RTFSSYAMA 1645 AAIKWSGTNT 1684 CAASMEAMNSLRVN 1834 YYA KERYYQSW 001S-G10 LRP6e1e2 RTFSRYVMG 1644 AAITWRGGST 1706 CATGPNSIY 1987 YYA 001S-A11 LRP6e1e2 RTFGNYDMG 1630 AGIRWSGSTL 1709 CYARTVIGGFGAFRA 2062 YA HW 001S-B11 LRP6e1e2 RRFTTYGMG 1623 AAVTWRSGST 1708 CAAGSTVVAEFNYW 1828 YYA 001S-C11 LRP6e1e2 SISSFNTMG 1659 AVITTGGDTSYS 1741 CNKVNAITKL 2025 001S-E11 LRP6e1e2 RTLSRYSMG 1651 AAISRSGDRIY 1697 CTLVNEIKTWW 2040 YS 001S-F11 LRP6e1e2 RTFSSYAMS 1646 AVIGRSGGIKY 1736 CATRRPFNSYNTEQS 1989 YA YDSW 001S-G11 LRP6e1e2 SIFRLGTMY 1655 ASIGKSGSTNYA 1719 CKQHPNGYR 2005 001S-H11 LRP6e1e2 RTLSSFAMG 1652 ATISRSGGNTY 1732 CNLREWNNSGAGY 2026 YA W 001S-A12 LRP6e1e2 IAFRYYDMG 1608 AAITWNGRSS 1704 CAAVFTGRFYGRPPR 1838 DYA EKYDYW 001S-B12 LRP6e1e2 RLLSYYALA 1622 AAISRNGDKS 1694 CTLVNEIKTWW 2038 HYS 001S-C12 LRP6e1e2 RTFSNYAVG 1641 AAISRFGGSTY 1693 CAADRIENYLGRYYD 1824 YV PSEYEYW 001S-D12 LRP6e1e2 RFFSRYAMG 1643 GAISRSGNNT 1744 CTLVNEIKTWW 2041 YYA 001S-F12 LRP6e1e2 RTFRSYTMG 1637 AAISGSGGSTT 1690 CNADIKTTTYSPLRNYW 2009 YA 008S-B01 LRP5 TIFSINTMG 1664 ATMTSGGNT 1734 CYRRQWASSWGAR 2064 NYA NYEYW 008S-C01 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CHGRDYGSNAPQYW 2001 MKYA 008S-D01 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CYVKLRDDDYVYR 2065 MKYA 008S-E01 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CNAVTYNGYTIR 2023 MKYA 008S-G01 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CYARTQRMGVVNSYW 2060 MKYA 008S-A02 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CNAVTFGGNTIR 2021 MKYA 008S-C02 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CNAVTYDGY 2022 MKYA 008S-D02 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CAAQFRNDYGLRYQ 1832 MKYA STNNYW 008S-E02 LRP5 NINSIETLG 1617 ANMRGGGY 1716 CNANYRGNRYW 2019 MKYA 009S-C01 LRP6e3e4 GSFSGYYWT 1595 GEINHSGATN 1745 CVRYAWPEFDHW 2053 RASQRVSNY 2089 AASSLQG 2110 CQQSYSVPYTF 2178 YN LN 009S-B02 LRP6e3e4 GSLSGYYWS 1596 GEINHSGSTNYN 1746 CVRYAWPEFDHW 2055 RASQSISNYLN 2090 AASSLQS 2110 CQQSYSLPLTF 2170 009S-C02 LRP6e3e4 GSFSDYYWS 1594 GEINHSGSTNYN 1746 CVRYAWPEFDHW 2054 RASQSISNYLN 2090 AASSLQS 2110 CQQSYSMPLTF 2171 009S-D02 LRP6e3e4 GTFSSYAIS 1603 GGIIPIFGTANYA 1749 CVYGRDFDYW 2056 SGSSSNVGN 2105 DNDKRPS 2122 CESWDSSLSSE 2139 NYVS VF 010S-A02 LRP6e1e2 HTFSSYAMG 1607 AAISQSGYVRYYA 1691 CKIYGLNGQPLGSW 2003 010S-B02 LRP6e1e2 RTFNSGTMG 1634 AAITWRGGIT 1705 CNADGYSWDGRSG 2008 YYA RRLELW 010S-D02 LRP6e1e2 RTFSSYAVG 1647 AAISYSGGSTK 1702 CAASVYISRRDSDYG 1837 YA YW 010S-E02 LRP6e1e2 LSSGRPFSSY 1612 AAISWSGGST 1701 CKLQVRPIGYSSAYSR 2004 VMG KYA NYW 010S-F02 LRP6e1e2 RSFNSYVIG 1625 AAIRWSGDNT 1688 CAASMEAMNSLRVN 1835 YYA KERYYQSW 009S-E02 LRP6e1e2 RRFTTYGMG 1623 AAVTWRSGST 1708 CAAGSTVVAEFNYW 1829 YYA 009S-F02 LRP6e1e2 RTFSYYAMG 1649 AAISRSGGIYYA 1698 CNTVRPLWAW 2029 009S-G02 LRP6e1e2 SIFSIYAMG 1658 AVITSGGKTVYA 1740 CYADSRSSWYDEYLE 2058 HW 009S-H02 LRP6e1e2 SIVRSLPMA 1660 ATINDAQRYYA 1727 CNTSPYMHDVW 2027 009S-A03 LRP6e1e2 RTFSVYGVG 1648 AAVSASGGYT 1707 CKAAPRWGGATAYW 2002 WYA 010S-G02 LRP6e1e2 SIVRSLPMA 1660 ATINDAQRYYA 1727 CNTSPYMHDVW 2028 010S-A03 LRP6e1e2 RTFRRYAMG 1636 ATISASGGNTA 1731 CNAPAWLYDDDYW 2020 YA 009S-B03 LRP6e1e2 RTFSNYAVG 1641 AAISRFGGSTY 1693 CAADRIENYLGRYYD 1825 YV PSEYEYW 010S-B03 LRP6e1e2 RTFSNYAVG 1641 AAISRFGGSTY 1692 CHAKQLRNGQMYTYW 1999 YA 010S-D03 LRP6e1e2 ISSVYGMG 1609 AAIQWSADNT 1686 CAARTSGGLFHYRRS 1833 FYA DHWDTW 009S-C03 LRP6e1e2 LPFSRYAMA 1610 AGMSGEGRN 1713 CSSRGYW 2034 TKYR 009S-D03 LRP6e1e2 SIFSDGAMG 1656 AVISGGRTGYA 1737 CNTYPFPIYKKGYPFW 2030 009S-E03 LRP6e1e2 RRFTTYGMG 1623 AAVTWRSGST 1708 CAAGSTVVAEFNYW 1830 YYA 009S-F03 LRP6e1e2 RTFSSYAMS 1646 AVIGRSGGIKY 1736 CATRRPFNSYNTEQS 1990 YA YDSW 010S-E03 LRP6e1e2 RSVSIYPMG 1628 AAINWSGDST 1685 CNAVVVGLSRRIDNIW 2024 KYA 010S-F03 LRP6e1e2 RTFSRYVMG 1644 AAITWRGGST 1706 CATGPNSIY 1988 YYA 009S-G03 LRP6e1e2 RSVSSYNMG 1629 AAISRRGGIIEYG 1695 CHAVENILGRFVDYW 2000 009S-H03 LRP6e1e2 SIFSINTMG 1657 AVITSGGKTVYA 1740 CYADSRSSWYDEYLE 2057 HW 009S-A04 LRP6e1e2 RTLSAYDMG 1650 GGIRWSGGTT 1753 CYARTVIGGFGAFRA 2063 LYP HW 009S-B04 LRP6e3e4 SIFMINTMA 1654 ATIRPVVSETT 1728 CNAKRPWGTRDEYW 2018 YA 010S-G03 LRP6e3e4 RSFNSYTTT 1624 AAIRGSSGSTF 1687 CNAASTVTAWPYYG 2006 YA PDYW 009S-C04 LRP6e3e4 FRFSISTMG 1553 AYITGGGRTM 1743 CNAFVRSDFDRYYDYW 2011 DG 009S-D04 LRP6e3e4 TIVSIYRIN 1665 AGITSSGRTIYA 1712 CNAASTVTAWPYYG 2007 PDYW 010S-H03 LRP6e3e4 RIFSIYDMG 1621 SGIRWSGGTS 1789 CSSRGYW 2035 YA 009S-E04 LRP6e3e4 RIFAIYDIA 1618 AMIRPVVTEID 1715 CNAKRPWGSRDEYW 2012 YA 010S-A04 LRP6e3e4 SLFSFNAVG 1662 ASISSGGRTNYA 1722 CSKGGVYGGTYVPD 2032 SW 009S-F04 LRP6e3e4 RSLSSFAMG 1627 ARISRGDGYT 1718 CAAVQAVIGGTLTTA 1839 DEA YDYW 010S-B04 LRP6e3e4 RVLSYYAMA 1653 AGITRGGATTY 1711 CAAGPNWSTRNREY 1827 YS DYW 009S-G04 LRP6e3e4 GTFSRYHMG 1601 SAITWSGGRT 1788 CALTWAPTPTNRRS 1872 YYA DYAYW 009S-H04 LRP6e3e4 RIFAIYDMA 1619 ATIRPVVSETT 1728 CNAKRPWGTRDEYW 2017 YA 010S-C04 LRP6e3e4 SLFSFNAMG 1661 ASISSGSRTNYA 1723 CSKGGVYGGTYVPD 2033 SW 010S-D04 LRP6e3e4 RIFAIYDIA 1618 ATIRPVVTQID 1730 CNAKRPWGSRDEYW 2015 YA 010S-E04 LRP6e3e4 RTFGSDVMG 1631 ALTGWGDGS 1714 CAAARRSGTYDIGQY 1820 TTYYE LRESAYVFW 010S-F04 LRP6e3e4 RTFSRYAMG 1643 AAITRSGSNTY 1703 CAADPRGVTLPRATA 1823 YA YEYW 009S-A05 LRP6e3e4 RTFSDYSMG 1639 AGISWIADNR 1710 CTAGRSRYLYGSSLN 2036 YYA GPYDYW 010S-G04 LRP6e3e4 VIFALYDIA 1666 ATIRPVVTETD 1729 CNAKRPWGSRDEYW 2014 YA 010S-H04 LRP6e3e4 RSFSDFFMG 1626 ATISWSGSSA 1733 CAAAYSYSQYGSSYS 1821 NYE YW 010S-A05 LRP6e3e4 LSFSSYAMG 1611 AAISRSGVSTY 1699 CAAKFGVLATTESRH 1831 YA DYW 010S-C05 LRP6e3e4 RTFNIDDMG 1633 ASIRWSGQSP 1720 CNAETYSGNTIW 2010 YYA 010S-D05 LRP6e3e4 RTFSDYSMA 1638 AGISWIADNR 1710 CAGDRSRYLYGDSLR 1841 YYA GPYGYW 010S-E05 LRP6e3e4 SVFTTFAKG 1663 ASITASSDRTF 1725 CAAYSTFNTDVASM 1840 YA KPDYW 010S-F05 LRP6e3e4 RIFSIYDIA 1620 ATIRPVVTETD 1729 CNAKRPWGSRDEYW 2013 YA 013S-G04 LRP6e3e4 RIFAIYDIA 1618 ATIRPVVSETT 1728 CNAKRPWGTRDEYW 2016 YA 013S-H04 LRP6e3e4 RFSMYDMG 1640 ASIRWSSGNT 1721 CYANIYYTRRAPEEYW 2059 WYA 013S-A05 LRP6e3e4 RTFNTYAMG 1635 ASVSWRYDRT 1726 CAADTNWRAGPRV 1826 YYT GIDEYAYW 013S-B05 LRP6e3e4 FAFSTTAMS 1549 STINPGGLSKS 1806 CTKGGIQ 2037 YA 013S-C05 LRP6e3e4 NIFPIDDMS 1616 ATVTSGGRINYA 1735 CNVDRTLYGKYKEYW 2031 013S-D05 LRP6e3e4 RIFSIYDMG 1621 SGIRWSGGTS 1789 CGSRGYW 1998 YA 013S-E05 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTADYA 1748 CARDWELYGMDVW 1907 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 013S-F05 LRP6e3e4 GTFSSYAIS 1603 GIINPSGGSTSYA 1761 CARAGYYDSSGYYAF 1882 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DIW 013S-G05 LRP6e3e4 YTFTYRYLH 1681 GGVIPIFGTAD 1755 CASDIVVDDAFDTW 1969 RASQDISNY 2079 AASTLQS 2113 CQQGNSFPYTF 2152 YA LN 010S-G06 LRP6e3e4 FSFETYGMS 1555 SGISGSGGRTH 1792 CARDLDYW 1897 QASQDISNY 2077 AASSLQS 2110 CQQSYRIHWTF 2163 YA LN 009S-B05 LRP6e3e4 FTFDAYAMH 1560 STLSGDANNA 1811 CARGGSGWSNYYG 1931 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YYA MDVW 009S-C05 LRP6e3e4 YTFTYRYLH 1681 GRIIPVLKITNYA 1768 CAVVDDAFDIW 1996 009S-D05 LRP6e3e4 FTLRNHWLS 1591 SAISGSGGSTY 1786 CATRTGYSYGFNFW 1991 RASQSISSYLN 2904 AASSLQS 2110 CQQSYSTPLTF 2174 YA AFDIW 009S-E05 LRP6e3e4 YTFTNNFMH 1676 GHVDPGDGET 1756 CARDWGIAAAGDYY 1908 RASQGINSY 2081 DAKGLHP 2114 CQQSYSAPLSF 2166 IYA YYGMDVW LA 009S-F05 LRP6e3e4 FTFDDYGMS 1561 SAIGTGGGTYYA 1781 CARLGSYGSPYYYYG 1959 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW 009S-G05 LRP6e3e4 FTFSDYYMS 1568 SGVSWNGSRT 1799 CAKDSGLV 1852 QASQDISNY 2077 AASTLQR 2112 CQQSYSAPLTF 2167 HYA LN 009S-C06 LRP6e3e4 YTFASYDIH 1671 GWMNPNSG 1776 CARATGSGWYTDLG 1883 RASRNINRY 2099 AASSLLS 2109 CQQSYNVPFTF 2162 NTGYA YW LN 009S-D06 LRP6e3e4 FTFSSHSTH 1573 STISDTNSGTY 1807 CAKAQATGWSGYYT 1843 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 009S-E06 LRP6e3e4 FTFTDYGLH 1587 AVISYGGSNKY 1739 CASGYSYGLYYYGM 1973 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 009S-F06 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATEAALDAFDIW 1985 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-G06 LRP6e3e4 YIFTDYYMH 1669 GWINPNSGGT 1774 CARDFLGSTGDYW 1892 RASQNIGLY 2088 DASSLQR 2121 CQQSYSTPYTF 2176 NYA LN 009S-H06 LRP6e3e4 FTFSSSAMH 1574 SAIGTGGSTYYA 1783 CAKGGDYFYYYYGM 1856 RASQSISSYLN 2094 AASLQS 2110 CQQSYSTPLTF 2174 DVW 009S-A07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATAYGSSSLNIDYW 1980 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-B07 LRP6e3e4 YTFTGYYMH 1675 GWINPNSGGT 1774 CVKDGGSFPLAYAFD 2049 RASQSISSYLN 2904 AASLQS 2110 CQQSYSTPLTF 2174 NYA IW 009S-D07 LRP6e3e4 FPFRYYGMS 1551 ARIGWNGGSI 1717 CARDYSDRSGIDYW 1910 RSSQSLLHS 2102 LGSNRAS 2131 CMQATQFPLTF 2146 VYA NGYNYLD 009S-F07 LRP6e3e4 GTFSSYAIS 1603 GIINPSGGSTS 1761 CARAAGNFWSGYYT 1876 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 009S-G07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARGSYGMDVW 1947 RASQGISNY 2083 DASNLET 2115 CLQDFSFPWTF 2140 YA LA 009S-H07 LRP6e3e4 YTFTGYYMH 1675 GWMNPNSG 1776 CASSVVPAGPAGVY 1975 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYA AFDIW 009S-A08 LRP6e3e4 GTFSSHAIN 1602 GWISANNGN 1775 CARDQDYGDYGWY 1902 RASQGISNY 2083 GSSTLQS 2127 CQQTYSIPPTF 2181 TDYA YYGMDVW LA 011S-C01 LRP6e3e4 LTFTSHGMS 1615 SYVSDSGSSVY 1818 CARHPGSFGGYSYA 1956 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA WYYYYGMDVW 009S-C08 LRP6e3e4 FSFNTFGIH 1556 AVISYDGSNKY 1738 CAKSIAAAGTGYYG 1868 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 009S-D08 LRP6e3e4 YTFTSYDIN 1679 GGIIPIFGTAN 1749 CARGPYYFDYW 1939 RASQGISNN 2082 DASSLES 2120 CLQHNSYPFTF 2143 YA LN 011S-F01 LRP6e3e4 FSFSDYYMS 1558 SGISESGGRTY 1790 CASAADFDYW 1966 RASQDISNY 2079 AASSLQS 2110 CLQDYSYPRTF 2141 YA LN 009S-E08 LRP6e3e4 TGFTGYYIH 1668 GWMNPNSG 1776 CARGYGDYDLW 1951 QASQDISNY 2077 DASSLES 2120 CQQSYRYPTF 2165 NTGYA LN 009S-F08 LRP6e3e4 DTFANYGFS 1547 GXVNAGNGN 1777 CAKGWLDFDYW 1866 QASQDISNY 2077 DASSLES 2120 CQQSYSTSITF 2177 TTYA LN 009S-G08 LRP6e3e4 FTFSDFAMT 1566 SYISGDSGYTN 1813 CARLGSYPGPYYYYM 1961 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 009S-H08 LRP6e3e4 YTFTDYFMN 1673 GIINPSGDSTR 1758 CARDDGLGGMDVW 1888 QASQDISNY 2076 AASSLQS 2110 CQQSYSTPLTF 2174 FA LA 009S-A09 LRP6e3e4 YTFTYRYLH 1681 GRIIPILGSTNYA 1767 CTTDLWDYW 2047 QASQGITNY 2078 AASSLQS 2110 CLQDYTDPFTF 2142 LN 011S-F02 LRP6e3e4 FTFSTYGMH 1584 SSISVSSGTTH 1804 CARGGSGSYYYAFDIW 1929 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-G02 LRP6e3e4 YTFTSYAMN 1678 GGIIPIFGTAN 1749 CARDASGGSTGWYY 1886 RASQGISSYLA 2087 AASSLQS 2110 CQQAYSFPWTF 2150 YA FDSW 011S-A03 LRP6e3e4 FTFSSYWMH 1580 STISGSGGRTY 1808 CATSPYGVFTLDYW 1993 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-C03 LRP6e3e4 YTFSYRYLH 1672 GGIIPIFGTAN 1749 CASTVTTDAFDIW 1977 QASQDISNY 2077 DASSLES 2120 CQQSYSFPPFTF 2168 YA LN 011S-D03 LRP6e3e4 FSFDDYGMS 1554 SVISSGGTIYYA 1812 CARHLSSGYLSYYGM 1954 RASQSISSYLA 2093 AASTLQS 2113 CQQSYSTPLTF 2174 DVW 011S-F03 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1859 KSSQSVLYTT 2073 WASSRKS 2135 CQQYYSTPYTF 2189 YA YW TNRNHIA 011S-C04 LRP6e1e2 GTFNSNAIS 1598 GWMNPNSG 1776 CARDYYGSGSYNYG 1912 GASQSVPR 2066 GASQRAT 2124 CQQYHNWPP 2184 NTGYA MDVW NSLA EYTF 011S-D04 LRP6e1e2 YTFTSYDIN 1679 GIINPSGGSTS 1761 CAREAYYYYYGMDVW 1915 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-H04 LRP6e1e2 YIFTDYYMH 1669 GRIIPILGRANYA 1765 CARGGYSTLDYW 1932 QASQDISNY 2077 AASTLQS 2113 CQQSFSTPRTF 2156 LN 008S-F02 LRP5 YTFTNYCMH 1677 GIINPSDGSTS 1757 CAKDMVHLIVALAID 1851 RSSQSLLHS 2101 TLSYRAS 2134 CMQALEALFTF 2144 HA YW DGYTYLY 010S-C06 LRP6e1e2 FTFNSYSMD 1563 SSISPRGGSTY 1802 CAPYYYDKSAKPLRSY 1875 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDHW 010S-E06 LRP6e3e4 LTVSSNYMS 1615 SGISWNSGSIG 1796 CARGSDCSGGSCYYS 1944 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 010S-F06 LRP6e3e4 FTFSSSWMH 1575 SAIGTGGGTYYA 1781 CAREVAVKDYYYYY 1921 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW 010S-H06 LRP6e3e4 YTFTSYDIN 1679 GRIIPILGRTNYA 1766 CAREERGATGRAFDIW 1918 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 010S-A07 LRP6e3e4 FTFSSYAMH 1576 ASISSTSGSKYYA 1724 CAKTYYDFWSGYYTF 1870 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 DYW 010S-B07 LRP6e3e4 FTFSDYYMS 1568 SMISYNGGRA 1800 CARGNPYYFDYW 1937 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 FYA 010S-C07 LRP6e3e4 FTFSKTDMH 1569 STITTDSRGTY 1810 CAKGGDYYYYYYGM 1858 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 010S-D07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CANGLEDAYAFDIW 1873 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-D05 LRP6e3e4 FTLRNHWLS 1591 SAISGSGGSTY 1786 CATRTGYSYGFNFW 1992 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA AFDIW 009S-E05 LRP6e3e4 YTFTNNFMH 1676 GHVDPGDGET 1756 CARDWGIAAAGDYY 1909 RASQGINSY 2081 DAKGLHP 2114 CQQSYSAPLSF 2166 IYA YYGMDVW LA 010S-E07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CAKDDFSLYGMDVW 1845 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-F05 LRP6e3e4 FTFDDYGMS 1561 SAIGTGGGTYYA 1781 CARLGSYGSPYYYYG 1960 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW 010S-F07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARLDYGETEGNGDW 1958 RASQSVYSN 2097 DTSNRAT 2123 CQQYNNWPP 2185 YA LA ITF 010S-G07 LRP6e3e4 FTFSSYAMH 1576 STISGSGGSTY 1809 CARAGYGRYYYGMD 1880 RVSQGISSYLN 2103 AASSLQS 2110 CQQTYTIPFTF 2182 YA VW 009S-G05 LRP6e3e4 FTFSDYYMS 1568 SGVSWNGSRT 1799 CAKDSGLV 1853 QASQDISNY 2077 AASTLQR 2112 CQQSYSAPLTF 2167 HYA LN 010S-H07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARDDSMGAFDIW 1890 QASQDISNY 2077 GTSNLQS 2128 CQQSYSTPYTF 2176 YA LN 010S-A08 LRP6e3e4 HTFLTYDIN 1606 GRITPRLGIAN 1770 CASYFGVMDVW 1979 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-A07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATAYGSSSLNIDYW 1981 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-B07 LRP6e3e4 YTFTGYYMH 1675 GWINPNSGGT 1774 CVKDGGSFPLAYAFD 2050 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NYA IW 009S-B06 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CAPALTDAGSFDYW 1874 RVSQSISSYLN 2103 AASSLQS 2110 CQQSYSTPLTF 2174 YA 010S-B08 LRP6e3e4 YTFTYRYLH 1681 GGIIPVFGTAD 1751 CARDREQQILDYW 1904 RASQGISNN 2082 DASNLET 2115 CQQSYTSRLTF 2179 YA LN 010S-C08 LRP6e3e4 FTFSTFGMH 1582 STITSSGGSTYYA 1809 CARAGIAAAPGSRNY 1878 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YGMDVW 009S-C06 LRP6e3e4 YTFASYDIH 1671 GWMNPNSG 1776 CARATGSGWYTDLG 1884 RASRNINRY 2099 AASSLLS 2109 CQQSYNVPFTF 2162 NTGYA YW LN 009S-D06 LRP6e3e4 FTFSSHSTH 1573 STISDTNSGTY 1807 CAKAQATGWSGYYT 1844 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 010S-D08 LRP6e3e4 FTFSSSWMH 1575 SAIGTGGGTYYA 1781 CAKEDYDSSGYYYYY 1855 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 FQHW 009S-E06 LRP6e3e4 FTFTDYGLH 1587 AVISYGGSNKY 1739 CASGYSYGLYYYGM 1974 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 010S-E08 LRP6e3e4 YSFTRTDMH 1670 GYISAYTGHTS 1778 CARDLGGTADYW 1898 RASQSISSYLN 2094 ZASSLQS 2137 CQQSYSTPLTF 2174 YA 010S-F08 LRP6e3e4 LTFDDHAMH 1613 SYISSSGRTIFYA 1815 CVRGDSGWGILYYV 2052 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW 009S-F06 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATEAALDAFDIW 1986 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 010S-G08 LRP6e3e4 YIFTDYYMH 1669 GGFDPEDGET 1747 CARGGGPNEHDYYF 1927 RASQSVRSS 2096 GSSSRAT 2126 CQQYGRSPRY 2183 IYA DYW DLA SF 010S-H08 LRP6e3e4 FTFZNAWMS 1590 SGISGSGGSTY 1793 CARGRGKKNYYYGM 1942 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 010S-A09 LRP6e3e4 FTFSTYYMS 1586 SGISWNGGKT 1794 CARGGDFDYW 1925 QASQDIANY 2075 AASSLQS 2110 CQQSYSTPYTF 2176 HYV LN 010S-B09 LRP6e3e4 GTFSSYAIS 1603 GWINPNSGDT 1773 CARGEQWLVWGFD 1924 RASQSISRYLN 2092 KASSLES 2130 CQQSYDSPWTF 2159 NYA PW 009S-G06 LRP6e3e4 YIFTDYYMH 1669 GWINPNSGGT 1774 CARDFLGSTGDYW 1893 RASQNIGLY 2088 DASSLQR 2121 CQQSYSTPYTF 2176 NYA LN 010S-C09 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARDEVEGGMDVW 1891 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-H06 LRP6e3e4 FTFSSSAMH 1574 SAIGTGGSTYYA 1783 CAKGGDYFYYYYGM 1857 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 DVW 010S-D09 LRP6e3e4 GTFSSYTIS 1603 GGIVPAYRRA 1754 CAKGGYELDYW 1865 QASQDISNY 2077 AASSLQS 2110 CQQIHSYPLTF 2155 NYA LN 010S-E09 LRP6e3e4 GDLSIYTIN 1593 GWINAGNGN 1772 CARGGDSSGYYYYAF 1926 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 TTYA DIW 009S-A07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATAYGSSSLNIDYW 1982 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-B07 LRP6e3e4 YTFTGYYMH 1675 GWINPNSGGT 1774 CVKDGGSFPLAYAFD 2051 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NYA IW 009S-D08 LRP6e3e4 YTFTSYDIN 1679 GGIIPIFGTAN 1749 CARGPYYFDYW 1940 RASQGISNNLN 2082 DASSLES 2120 CLQHNSYPFTF 2143 YA LN 010S-F09 LRP6e3e4 FTFDEYAMH 1562 STISGSGGSTY 1809 CASAKNDFWSGYFA 1968 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 010S-G09 LRP6e3e4 GTFNTHTIT 1599 GWMNPNSG 1776 CARGNLDFDYW 1936 QASQDISNY 2077 DASNLET 2115 CQQSYSTPLTF 2174 NTGYA LN 010S-H09 LRP6e3e4 FTFSDHYMS 1567 SAISSGSDRTY 1787 CARYSGYDFDYW 1965 RASQGISNY 2084 AASTLQS 2113 CQQGYGTPP 2153 YA LN MF 010S-A10 LRP6e3e4 FSFSSYSMN 1559 SYISSSSSTIYYA 1816 CARGSGYYGPGYYG 1946 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 MDVW 009S-D07 LRP6e3e4 FPFRYYGMS 1551 ARIGWNGGSI 1717 CARDYSDRSGIDYW 1911 RSSQSLLHS 2102 LGSNRAS 2131 CMQATQFPLTF 2146 VYA NGYNYLD 010S-B10 LRP6e3e4 FAFKDYYMT 1548 SAIGAGGGTY 1779 CARESALYSSSWYYY 1920 RASQSISSYLN 2094 GTSSLHT 2129 CQQANSFPFTF 2148 YA YYGMDVW 010S-C10 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1860 KSSQSILSSS 2072 WASSRKS 2135 CQQYYNIPYSF 2187 YA YW SNRDSLA 009S-E07 LRP6e3e4 YTFTGYYIH 1674 ZHVDPEDGETI 1819 CARGPAAIGILGWFD 1938 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA PW 010S-D10 LRP6e3e4 YIFTDYYMH 1669 GWMNPNSG 1776 CARTLSGYSSSWYVF 1964 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYA DYW 010S-E10 LRP6e3e4 FTFSSYSMN 1579 SGISWNSGTT 1797 CARDHSSGWRHYFD 1895 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 GYS YW 010S-F10 LRP6e3e4 FTFSNSDMN 1570 SYISGNSGYTN 1814 CASGSYYSDFDYW 1971 RASQSISNYLN 2090 AASTLES 2111 CQQANSFPPTF 2148 YA 010S-G10 LRP6e3e4 GTFSSYAIS 1603 GRINPNGGGT 1769 CAREGGYYFDYW 1919 RASQGISNY 2083 AASSLQS 2110 CQQSYSTPWTF 2175 IYA LA 009S-F07 LRP6e3e4 GTFSSYAIS 1603 GIINPSGGSTS 1761 CARAAGNFWSGYYT 1877 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA FDYW 009S-G07 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARGSYGMDVW 1948 RASQGISNY 2083 DASNLET 2115 CLQDFSFPWTF 2140 YA LA 010S-H10 LRP6e3e4 YTFTSYYMH 1680 GWINPNSGGT 1774 CAREAAEIPVGAFDIW 1914 KSSHSLLYSS 2071 WSSTRES 2136 CQQYYSTPQTF 2188 NYA DNKNYLA 010S-A11 LRP6e3e4 FTFSNSDMN 1570 SYISGNSGYTN 1814 CASGSYYSDFDYW 1972 RASQSIZNYLN 2095 ZASTLES 2138 CQQANSFPPTF 2148 YA 010S-B11 LRP6e3e4 FTFRNYAIH 1564 SAIGTGGDTYYA 1780 CARDGGIRDFDYW 1894 QASQDISNY 2077 AASTLQS 2113 CQQSYSTPLTF 2174 LN 010S-C11 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CAADDLGLELHYW 1822 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-H07 LRP6e3e4 YTFTGYYMH 1675 GWMNPNSG 1776 CASSVVPAGPAGVY 1976 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYA AFDIW 009S-A08 LRP6e3e4 GTFSSHAIN 1602 GWISANNGN 1775 CARDQDYGDYGWY 1903 RASQGISNY 2083 GSSTLQS 2127 CQQTYSIPPTF 2181 TDYA YYGMDVW LA 010S-D11 LRP6e3e4 YTFTYRYLH 1681 GGIIPVFGTAN 1752 CATDEYSSSYAFDIW 1983 RASQSVSSN 2097 GASTRAT 2125 CQQFDRSPLTF 2151 YA LA 010S-E11 LRP6e3e4 FTFSAHGMH 1565 SGISESGGSTY 1791 CARGRGYSYGYYAFD 1943 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA IW 010S-F11 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CARDSDWGVVDPW 1905 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 010S-G11 LRP6e3e4 YTFTYRYLH 1681 GRIIPVLKITNYA 1768 CAVVDDAFDIW 1997 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 010S-H11 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CAKDGTDGRFDPW 1846 RASQDISSYLA 2080 SASTLQS 2133 CQQSNSFPYTF 2157 YA 009S-B08 LRP6e3e4 FTFTSSAVQ 1589 GWINAGNGN 1772 CARRGGDVTVPAAY 1963 RASQSISSYLN 2094 ZASSLQS 2137 CQQSYSTPLTF 2174 TTYA YAMDVW 010S-A12 LRP6e3e4 VTFSRYPIS 1667 GGIIPIFGTAN 1749 CAKDSGNYGYYGMD 1854 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA VW 010S-B12 LRP6e3e4 FTFSSYDMH 1578 SGITSNGGATY 1798 CARGTTGKGYYYYG 1949 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 010S-C12 LRP6e3e4 FTFSNYWIH 1571 SAIGTGGGTYYA 1781 CTTAGYKAARRSVYP 2044 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 RIFNFDYW 010S-D12 LRP6e3e4 YTFTYRYLH 1681 GRIIPIFGTANYA 1763 CAREEGVGGMDVW 1917 PRSQSIGSW 2100 DASNLQS 2116 CQQSSSTPYTF 2158 LA 010S-E12 LRP6e3e4 FTFSSYAMH 1576 SAIGAGGGTY 1779 CARGVSSGYYYYYG 1950 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 010S-F12 LRP6e3e4 FTVSSNYMS 1592 SAIGTGGGTYYA 1781 CARAGTNWGGWYF 1879 RASQGISRD 2085 AASTLQS 2113 CQQSYSPPFTF 2172 DLW LA 010S-G12 LRP6e3e4 FALSGYYMS 1550 SSISSSSTYIRYA 1803 CATVTGYSSAGAFDIW 1995 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 011S-A01 LRP6e3e4 FTFSTHAFH 1583 SAIRGSGERTY 1784 CARDLRNWGSPYW 1901 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA YFDLW 011S-B01 LRP6e3e4 GTFSHYTIS 1600 GWINAGNGN 1771 CAKGGSLDMDVW 1864 RASQGISNY 2083 AASSLHS 2108 CQQSYRTPLTF 2164 TKYS LA 011S-C01 LRP6e3e4 LTFTSHGMS 1615 SYVSDSGSSVY 1818 CARHPGSFGGYSYA 1957 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA WYYYYGMDVW 011S-D01 LRP6e3e4 GTISDYTVS 1605 GIINPSGGSTS 1761 CARGYYDFDYW 1953 RASQGISNY 2083 AASSLQS 2110 CQQSYSTPLTF 2174 YA LA 009S-C08 LRP6e3e4 FSFNTFGIH 1556 AVISYDGSNKY 1738 CAKSIAAAGTGYYG 1869 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 011S-E01 LRP6e3e4 FPFZYYSMN 1552 SAISGRDGRTY 1785 CAKDLGIQLPDYYFD 1847 RASQGISSA 2086 AASTLQS 2113 CQQSYSSPPTF 2173 YA YW LA 009S-D08 LRP6e3e4 YTFTSYDIN 1679 GGIIPIFGTAN 1749 CARGPYYFDYW 1941 RASQGISNN 2082 DASSLES 2120 CLQHNSYPFTF 2143 YA LN 011S-F01 LRP6e3e4 FSFSDYYMS 1558 SGISESGGRTY 1790 CASAADFDYW 1967 RASQDISNY 2079 AASSLQS 2110 CLQDYSYPRTF 2141 YA LN 009S-E08 LRP6e3e4 YGFTGYYIH 1668 GWMNPNSG 1776 CARGYGDYDLW 1952 QASQDISNY 2077 DASSLES 2120 CQQSYRYPTF 2165 NTGYA LN 009S-F08 LRP6e3e4 DTFANYGFS 1547 GXVNAGNGN 1777 CAKGWLDFDYW 1867 QASQDISNY 2077 DASSLES 2120 CQQSYSTSITF 2177 TTYA LN 011S-G01 LRP6e3e4 YTFTYRYLH 1681 GGIIPLFGTAN 1750 CTTDDYGDQYGMD 2046 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA VW 011S-H01 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CTTDDYGDLTHLDYW 2045 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-A02 LRP6e3e4 GTFSSYAIS 1603 GWMNPNSG 1776 CARDKGYAFDIW 1896 RSSQSLLHS 2102 AASSLQS 2110 CMQALQTPITF 2145 NTGYA NGYNYLD 011S-B02 LRP6e3e4 YSFTRTDMH 1670 GYISAYTGHTS 1778 CARDLGGTADYW 1899 RZSQSZSZYLN 2104 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-C02 LRP6e3e4 FTFSTYSMN 1585 SGISWNSGRI 1795 CARDVGAFDIW 1906 QASQDISNY 2077 AASILQS 2107 CQQSYSIPFTF 2169 GYA LN 009S-G08 LRP6e3e4 FTFSDFAMT 1566 SYISGDSGYTN 1813 CARLGSYPGPYYYYM 1962 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 011S-D02 LRP6e3e4 FTFSSYAMS 1577 SSISGSGGVTY 1801 CARGGNTYYYYYGM 1928 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA DVW 009S-H08 LRP6e3e4 YTFTDYFMN 1673 GIINPSGDSTR 1758 CARDDGLGGMDVW 1889 QASQDISNY 2076 AASSLQS 2110 CQQSYSTPLTF 2174 FA LA 011S-E02 LRP6e3e4 YTFTYRYLH 1681 GGIIPIFGTAN 1749 CATDYGDYYYGMDVW 1984 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 009S-A09 LRP6e3e4 YTFTYRYLH 1681 GRIIPILGSTNYA 1767 CTTDLWDYW 2048 QASQGITNY 2078 AASSLQS 2110 CLQDYTDPFTF 2142 LN 011S-F02 LRP6e3e4 FTFSTYGMH 1584 SSISVSSGTTH 1805 CARGGSGSYYYAFDIW 1930 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-G02 LRP6e3e4 YTFTSYAMN 1678 GGIIPIFGTAN 1749 CARDASGGSTGWYY 1887 RASQGISSYLA 2087 AASSLQS 2110 CQQAYSFPWTF 2150 YA FDSW 011S-H02 LRP6e3e4 YTFTNNFMH 1676 GIINPSGGSTS 1761 CARGLYKRYSYGYG 1935 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 009S-B09 LRP6e3e4 FSFNTYAMN 1557 AVTSYDGGKK 1742 CARDAGGDYDYW 1885 QASQDISNY 2077 AASSLQS 2110 CQQSYNTPRTF 2161 NYA LN 009S-C09 LRP6e3e4 GTFHTYGLS 1597 GGIIPIFGTAN 1749 CARGSGWSGLDYW 1945 QASQDISNY 2077 DASNLET 2115 CQQSYTTPFTF 2180 YA LN 011S-A03 LRP6e3e4 FTFSSYWMH 1580 STISGSGGRTY 1808 CATSPYGVFTLDYW 1994 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-B03 LRP6e3e4 GTFSZYAIS 1604 GIINPSGGSTN 1760 CARAGYWSGYGYYG 1881 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA MDVW 011S-C03 LRP6e3e4 YTFSYRYLH 1672 GGIIPIFGTAN 1749 CASTVTTDAFDIW 1978 QASQDISNY 2077 DASSLES 2120 CQQSYSFPPFTF 2168 YA LN 011S-D03 LRP6e3e4 FSFDDYGMS 1554 SVISSGGTIYYA 1812 CARHLSSGYLSYYGM 1955 RASQSISSYLA 2093 AASTLQS 2113 CQQSYSTPLTF 2174 DVW 009S-F09 LRP6e3e4 YSFTRTDMH 1670 GYISAYTGHTS 1778 CARDLGGTADYW 1900 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-E03 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1861 KSSHSLLSTS 2070 WASSRKS 2135 CQQYNNWPY 2186 YA YW TNRNQLA TF 009S-G09 LRP6e3e4 FTFSRHSMN 1572 SYSSGNSGYT 1817 CARGDLEFDYW 1923 RASQGISNY 2083 SASSLQS 2132 CQQGYNTPRTF 2154 NYA LA 011S-F03 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1862 KSSQSVLYTT 2074 WASSRKS 2135 CQQYYSTPYTF 2189 YA YW TNRNHIA 009S-H09 LRP6e3e4 FTFSSYAMS 1577 SAISGSGGSTY 1786 CAKGGRDGYKGYFD 1863 KSSHSLLSTS 2069 WASSRKS 2135 CQQYYNIPYSF 2187 YA YW TNRNHLA 011S-G03 LRP6e3e4 YTFTYRYLH 1681 GRIIPIHGIANYA 1764 CAREYSYGYFRYW 1922 RASQGISSYLA 2087 DASNLET 2115 CQQANSLFTF 2149 009S-A10 LRP6e3e4 FTFTSSAMQ 1588 GIINPSGGSTIYA 1759 CASGDTYDLYSLDVW 1970 RASQSISRW 2091 AASSLQS 2110 CQQAYSFPWTF 2150 LA 009S-B10 LRP6e3e4 YIFTDYYMH 1669 GWINAGNGN 1772 CAKVASGWSWPFDIW 1871 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 TTYA 011S-B04 LRP6e1e2 YTFTSYDIN 1679 GIINPSGGSTS 1761 CTREHSYYYYGMDVW 2042 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-C04 LRP6e1e2 GTFNSNAIS 1598 GWMNPNSG 1776 CARDYYGSGSYNYG 1913 GASQSVPR 2066 GASQRAT 2124 CQQYHNWPP 2184 NTGYA MDVW NSLA EYTF 011S-D04 LRP6e1e2 YTFTSYDIN 1679 GIINPSGGSTS 1761 CAREAYYYYYGMDVW 1916 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 YA 011S-E04 LRP6e1e2 FTFSSZZMH 1581 SAIGTGGGTZYA 1782 CAKDLGRAAAGSMD 1850 WASQSVRG 2106 DASNRAA 2117 CQHRSNWPLTF 2147 VW NYVA 011S-F04 LRP6e1e2 YIFTDYYMH 1669 GRIIPILGRANYA 1765 CARGGYSTLDYW 1933 HGSQDISNY 2067 DASNRQS 2119 CQQSFSTPRTF 2156 LN 011S-H04 LRP6e1e2 YIFTDYYMH 1669 GRIIPILGRANYA 1765 CARGGYSTLDYW 1934 QASQDISNY 2077 AASTLQS 2113 CQQSFSTPRTF 2156 LN 011S-A05 LRP6e1e2 FTFSSYAMH 1576 SAIGTGGGTYYA 1781 CAKDLGRAAAGSMD 1848 WASQSVRG 2106 DASNRAG 2118 CQHRSNWPLTF 2147 VW NYVA 011S-B05 LRP6e1e2 YZFTDYYMH 1683 GWMNPNSG 1776 CTRVAWGLDYW 2043 RASQSISSYLN 2094 AASSLQS 2110 CQQSYSTPLTF 2174 NTGYA 011S-C05 LRP6e1e2 FTFSSYAMH 1576 SAIGTGGGTYYA 1781 CAKDLGRAAAGSMD 1849 WASQSVRG 2106 DASNRAA 2117 CQHRSNWPLTF 2147 VW NYVA 1115.3 LRP6e1e2 GFSFSTS NLNGGS ELAGYGTPFAY KASQSISYNLH YTSQSIS QQSNSWPLT  421.1 LRP6e1e2 GYTFTTY FPGNVNT EELQYYFDY SANSSVRFMF FTSNLAS QQYHSYPWT YW211.31. LRP6e3e4 GFTFTSY SPYSGS RARPPIRLHPRGSVM RASQDVSTA SASFLYS QQSYTTPPT 57 DY VA

TABLE 2B Anti-LRP5/6 Antibody Clone IDs, Heavy Chain (HC) Seq ID Nos, and Binding Characteristics. Clone ID HC Seq ID NO Confirmed Binding 001S-F11 66 LRP6e1e2 009S-G02 67 LRP6e1e2 009S-A03 68 LRP6e1e2 009S-D03 69 LRP6e1e2 009S-F03 70 LRP6e1e2 009S-H03 71 LRP6e1e2 009S-A04 72 LRP6e1e2 009S-B04 73 LRP6e3e4 009S-D04 74 LRP6e3e4 009S-E04 75 LRP6e3e4 009S-F04 76 LRP6e3e4 009S-G04 77 LRP6e3e4 009S-H04 78 LRP6e3e4 009S-A05 79 LRP6e3e4 013S-G04 80 LRP6e3e4 013S-H04 81 LRP6e3e4 013S-C05 82 LRP6e3e4 013S-D05 83 LRP6e3e4 013S-G04 84 LRP6e3e4 013S-H04 85 LRP6e3e4 013S-A05 86 LRP6e3e4 013S-C05 87 LRP6e3e4 013S-D05 88 LRP6e3e4 008S-D01 133 LRP5

In certain embodiment, the LRP5/6 binding domain may be selected from any binding domain that binds LRP5 or LRP6 with a K_(D) of less than or equal to about 1×10⁻⁴ M, less than or equal to about 1×10⁻⁵ M, less than or equal to about 1×10⁻⁶ M, less than or equal to about 1×10⁻⁷ M, less than or equal to about 1×10⁻⁸ M, less than or equal to about 1×10⁻⁹ M, or less than or equal to about 1×10⁻¹⁰ M in the context of a Wnt surrogate molecule. In certain embodiment, the LRP5/6 binding domain may be selected from any binding domain that binds LRP5 or LRP6 with a K_(D) of greater than or equal to about 1×10⁻⁴ M, greater than or equal to about 1×10⁻⁵ M, greater than or equal to about 1×10⁻⁶ M, greater than or equal to about 1×10⁻⁷ M, greater than or equal to about 1×10⁻⁸ M, greater than or equal to about 1×10⁻⁹ M, or greater than about 1×10⁻¹⁰ M in the context of a Wnt surrogate molecule. In certain embodiment, the LRP5/6 binding domain may be selected from any binding domain that binds LRP5 or LRP6 at high affinity, e.g. a K_(D) of less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, less than about 1×10⁻⁹ M, or less than about 1×10⁻¹⁰ M.

Other suitable LRP5/6 binding domains include, without limitation, de novo designed LRP5/6 binding proteins, antibody derived binding proteins, e.g. scFv, Fab, etc. and other portions of antibodies that specifically bind to one or more Fzd proteins; VHH or sdAb derived binding domains; knottin-based engineered scaffolds; naturally occurring LRP5/6, including without limitation, DKK1, DKK2, DKK3, DKK4, sclerostin; Wise; fusions proteins comprising any of the above; derivatives of any of the above; variants of any of the above; and biologically active fragments of any of the above, and the like. A LRP5/6 binding domain may be affinity selected to enhance binding.

Members of the Dickkopf (DKK) gene family (see Krupnik et al. (1999) Gene 238(2):301-13) include DKK-1, DKK-2, DKK-3, and DKK-4, and the DKK-3 related protein Soggy (Sgy). hDKKs 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Exemplary sequences of human Dkk genes and proteins are publicly available, e.g., Genbank accession number NM_014419 (soggy-1); NM_014420 (DKK4); AF177394 (DKK-1); AF177395 (DKK-2); NM_015881 (DKK3); and NM_014421 (DKK2). In some embodiments of the invention, the Lrp6 binding moiety is a DKK1 peptide, including without limitation the C-terminal domain of human DKK1. The C-terminal domain may comprise the sequence:

(SEQ ID NO: 2190) KMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKH RRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH (see Genbank accession number NP_036374) or a biologically active fragment thereof.

Binding of DKK proteins to LRP5/6 are discussed, for example in Brott and Sokol Mol. Cell. Biol. 22 (17), 6100-6110 (2002); and Li et al. J. Biol. Chem. 277 (8), 5977-5981 (2002), each herein specifically incorporated by reference. The corresponding region of human DKK2 (Genbank reference NP_055236) may comprise the sequence: KMSHIKGHEGDPCLRSSDCIEGFCCARHFWTKICKPVLHQGEVCTKQRKKGSH GLEIFQRCDCAKGLSCKVWKDATYSSKARLHVCQK (SEQ ID NO:2191) or a biologically active fragment thereof.

Antibodies that specifically bind to LRP5 or LRP6 are known in the art and are commercially available, or can be generated de novo. LRP5, LRP6 or fragments thereof can be used as an immunogen or in screening assays to develop an antibody. Examples of known antibodies include, without limitation, those described in Gong et al. (2010) PLoS One. 5(9):e12682; Ettenberg et al. (2010) Proc Natl Acad Sci USA. 107(35):15473-8; and those commercially available from, for example Santa Cruz biotechnology antibody clone 1A12, which was raised against synthetic LRP5/6 of human origin and binds to both the full length and proteolytic fragment of LRP6 and LRP5 of mouse and human origin; the monoclonal antibody 2611; Cell Signaling Technology antibody specific for LRP5 (D80F2), catalog number 5731; etc.

In certain embodiments, Wnt surrogate molecules disclosed herein comprise one or more polypeptides comprising two or more binding regions. For example, the two or more binding regions may be two or more Fzd binding regions or two or more LRP5/6 binding regions, or they may comprise one or more Fzd binding region and one or more LRP5/6 binding region. The binding regions may be directly joined or contiguous, or may be separated by a linker, e.g. a polypeptide linker, or a non-peptidic linker, etc. The length of the linker, and therefore the spacing between the binding domains can be used to modulate the signal strength, and can be selected depending on the desired use of the Wnt surrogate molecule. The enforced distance between binding domains can vary, but in certain embodiments may be less than about 100 angstroms, less than about 90 angstroms, less than about 80 angstroms, less than about 70 angstroms, less than about 60 angstroms, or less than about 50 angstroms. In some embodiments the linker is a rigid linker, in other embodiments the linker is a flexible linker. In certain embodiments where the linker is a peptide linker, it may be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids in length, and is of sufficient length and amino acid composition to enforce the distance between binding domains. In some embodiments, the linker comprises or consists of one or more glycine and/or serine residues.

In particular embodiments, a Wnt surrogate molecule comprises a polypeptide sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to a polypeptide sequence disclosed in any of SEQ ID NOs:89-128 or 134-157, or having at least 90%, at least 95%, at least 98% or at least 99% identity to an antigen-binding fragment of a polypeptide sequence disclosed in any of SEQ ID NOs:89-128 or 134-157. In certain embodiments, the Wnt surrogate molecules comprises or consists of a polypeptide sequence set forth in any of SEQ ID NOs:89-128 or 134-157, or an antigen-binding fragment thereof. In particular embodiments, the antigen-binding fragment binds one or more Fzd receptors and also binds LRP5 and/or LRP6.

Wnt surrogate molecule can be multimerized, e.g. through an Fc domain, by concatenation, coiled coils, polypeptide zippers, biotin/avidin or streptavidin multimerization, and the like. The Wnt surrogate molecules can also be joined to a moiety such as PEG, Fc, etc., as known in the art to enhance stability in vivo.

In certain embodiments, a Wnt surrogate molecule directly activates canonical Wnt signaling through binding to one or more Fzd proteins and to LRP5/6, particularly by binding to these proteins on a cell surface, e.g., the surface of a human cell. The direct activation of Wnt signaling by a Wnt surrogate molecule is in contrast to potentiation of Writ signaling, which enhances activity only when native Wnt proteins are present.

Wnt surrogate molecules may activate Wnt signaling, e.g., by mimicking the effect or activity of a Wnt protein binding to a frizzled protein. The ability of the Wnt surrogate molecules of the invention to mimic the activity of Wnt can be confirmed by a number of assays. The Wnt surrogate molecules typically initiate a reaction or activity that is similar to or the same as that initiated by the receptor's natural ligand. In particular, the Wnt surrogate molecules of the invention enhance the canonical Wnt/β-catenin signaling pathway. As used herein, the term “enhances” refers to a measurable increase in the level of Wnt/β-catenin signaling compared with the level in the absence of a Wnt surrogate molecule of the invention.

Various methods are known in the art for measuring the level of canonical Wnt/β-catenin signaling. These include, but are not limited to assays that measure: Wnt/β-catenin target gene expression; TCF reporter gene expression; β-catenin stabilization; LRP phosphorylation; Axin translocation from cytoplasm to cell membrane and binding to LRP. The canonical Wnt/β-catenin signaling pathway ultimately leads to changes in gene expression through the transcription factors TCF7, TCF7L1, TCF7L2 (a.k.a., TCF4), and LEF. The transcriptional response to Wnt activation has been characterized in a number of cells and tissues. As such, global transcriptional profiling by methods well known in the art can be used to assess Wnt/β-catenin signaling activation or inhibition.

Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factors. A TCF reporter assay assesses changes in the transcription of TCF/LEF controlled genes to determine the level of Wnt/β-catenin signaling. A TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP/FOP this method involves the use of three copies of the optimal TCF motif CCTTTGATC, or three copies of the mutant motif CCTTTGGCC, upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFI_ASH and pFOPFI_ASH, respectively) to determine the transactivational activity of endogenous p-catenin/TCF4. A higher ratio of these two reporter activities (TOP/FOP) indicates higher β-catenin/TCF4 activity, whereas a lower ratio of these two reporter activities indicates lower β-catenin/TCF4 activity.

Various other reporter transgenes that respond to Wnt signals exist intact in animals and therefore, effectively reflect endogenous Wnt signaling. These reporters are based on a multimerized TCF binding site, which drives expression of LacZ or GFP, which are readily detectable by methods known in the art. These reporter genes include: TOP-GAL, BAT-GAL, ins-TOPE FP, ins-TOPGAL, LEF-EGFP, Axin2-LacZ, Axin2-d2EGFP, Lgr5tm1 (cre/ERT2), TOPdGFP.

The recruitment of dephosphorylated β-catenin to the membrane, stabilization and phosphorylation status of β-catenin, and translocation of β-catenin to the nucleus (Klapholz-Brown Z et al., PLoS One. 2(9) e945, 2007), in some cases mediated by complex formation with TCF transcription factors and TNIK are key steps in the Writ signaling pathway. Stabilization is mediated by Disheveled family proteins that inhibit the “destruction” complex so that degradation of intracellular β-catenin is reduced, and translocation of β-catenin to the nucleus follows thereafter. Therefore, measuring the level and location of β-catenin in a cell is a good reflection of the level of Wnt/β-catenin signaling. A non-limiting example of such an assay is the “BioImage β-Catenin Redistribution Assay” (Thermo Scientific) which provides recombinant U20S cells that stably express human β-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). Imaging and analysis is performed with a fluorescence microscope or HCS platform allowing the levels and distribution of EGFP-β-catenin to be visualized.

Another way, in which the destruction complex is inhibited, is by removal of Axin by recruitment of Axin to the cytoplasmic tail of the Wnt co-receptor LRP. Axin has been shown to bind preferentially to a phosphorylated form of the LRP tail. Visualization of Axin translocation, for example with a GFP-Axin fusion protein, is therefore another method for assessing levels of Wnt/β-catenin signaling.

In certain embodiments, a Wnt surrogate molecule enhances or increases canonical Wnt pathway signaling, e.g., β-catenin signaling, by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 150%, 200%, 250%, 300%, 400% or 500%, as compared to the β-catenin signaling induced by a neutral substance or negative control as measured in an assay described above, for example as measured in the TOPFlash assay. A negative control may be included in these assays. In particular embodiments, Wnt surrogate molecules may enhance β-catenin signaling by a factor of 2×, 5×, 10×, 100×, 1000×, 10000× or more as compared to the activity in the absence of the Wnt surrogate molecule when measured in an assay described above, for example when measured in the TOPFlash assay, or any of the other assays mentioned herein.

“Wnt gene product” or “Wnt polypeptide” when used herein encompass native sequence Writ polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments and chimeric Wnt polypeptides. In particular embodiments, a Wnt polypeptide is a native human full length mature Wnt protein.

For example, human native sequence Wnt proteins of interest in the present application include the following: Wnt-1 (GenBank Accession No. NM_005430); Wnt-2 (GenBank Accession No. NM_003391); Wnt-2B (Wnt-13) (GenBank Accession No. NM_004185 (isoform 1), NM_024494.2 (isoform 2)), Wnt-3 (RefSeq.: NM_030753), Wnt3a (GenBank Accession No. NM_033131), Wnt-4 (GenBank Accession No. NM_030761), Wnt-5A (GenBank Accession No. NM_003392), Wnt-5B (GenBank Accession No. NM_032642), Wnt-6 (GenBank Accession No. NM_006522), Wnt-7A (GenBank Accession No. NM_004625), Wnt-7B (GenBank Accession No. NM_058238), Wnt-8A (GenBank Accession No. NM_058244), Wnt-8B (GenBank Accession No. NM_003393), Wnt-9A (Wnt-14) (GenBank Accession No. NM_003395), Wnt-9B (Wnt-15) (GenBank Accession No. NM_003396), Wnt-1 OA (GenBank Accession No. NM_025216), Wnt-10B (GenBank Accession No. NM_003394), Wnt-11 (GenBank Accession No. NM_004626), Wnt-16 (GenBank Accession No. NM_016087)). Although each member has varying degrees of sequence identity with the family, all encode small (i.e., 39-46 kD), acylated, palmitoylated, secreted glycoproteins that contain 23-24 conserved cysteine residues whose spacing is highly conserved (McMahon, A P et al., Trends Genet. 1992; 8: 236-242; Miller, J R. Genome Biol. 2002; 3(1): 3001.1-3001.15). Other native sequence Wnt polypeptides of interest include orthologs of the above from any mammal, including domestic and farm animals, and zoo, laboratory or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rats, mice, frogs, zebra fish, fruit fly, worm, etc.

“Wnt pathway signaling” or “Wnt signaling” is used herein to refer to the mechanism by which a biologically active Wnt exerts its effects upon a cell to modulate a cell's activity. Wnt proteins modulate cell activity by binding to Wnt receptors, including proteins from the Frizzled (Fzd) family of proteins, proteins from the ROR family of proteins, the proteins LRP5, LRP6 from the LRP family of proteins, the protein FRL1/crypto, and the protein Derailed/Ryk. Once activated by Wnt binding, the Wnt receptor(s) will activate one or more intracellular signaling cascades. These include the canonical Wnt signaling pathway; the Wnt/planar cell polarity (Wnt/PCP) pathway; the Wnt-calcium (Wnt/Ca²⁺) pathway (Giles, R H et al. (2003) Biochim Biophys Acta 1653, 1-24; Peifer, M. et al. (1994) Development 120: 369-380; Papkoff, J. et al (1996) Mol. Cell Biol. 16: 2128-2134; Veeman, M. T. et al. (2003) Dev. Cell 5: 367-377); and other Writ signaling pathways as is well known in the art.

For example, activation of the canonical Wnt signaling pathway results in the inhibition of phosphorylation of the intracellular protein β-catenin, leading to an accumulation of β-catenin in the cytosol and its subsequent translocation to the nucleus where it interacts with transcription factors, e.g. TCF/LEF, to activate target genes. Activation of the Wnt/PCP pathway activates RhoA, c-Jun N-terminal kinase (JNK), and nemo-like kinase (NLK) signaling cascades to control such biological processes as tissue polarity and cell movement. Activation of the Wnt/Ca²⁺ by, for example, binding of Wnt-4, Wnt-5A or Wnt-11, elicits an intracellular release of calcium ions, which activates calcium sensitive enzymes like protein kinase C (PKC), calcium-calmodulin dependent kinase II (CamKII) or calcineurin (CaCN). By assaying for activity of the above signaling pathways, the biological activity of an antibody or antigen-binding fragment thereof, e.g., a Wnt surrogate molecule, can be readily determined.

In certain embodiments, functional properties of Wnt surrogate molecules may be assessed using a variety of methods known to the skilled person, including e.g., affinity/binding assays (for example, surface plasmon resonance, competitive inhibition assays), cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays in response to a Wnt, cancer cell and/or tumor growth inhibition using in vitro or in vivo models, including but not limited to any described herein. The Wnt surrogate molecules described herein may also be tested for effects on Fzd receptor internalization, in vitro and in vivo efficacy, etc. Such assays may be performed using well-established protocols known to the skilled person (see e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); or commercially available kits.

In certain embodiments, a Fzd-binding region of a Wnt surrogate molecule (e.g., an antigen-binding fragment of an anti-Fzd antibody) comprises one or more of the CDRs of the anti-Fzd antibodies described herein. In certain embodiments, a LRP5/6-binding region of a Wnt surrogate molecule (e.g., an antigen-binding fragment of an anti-LRP5/6 antibody) comprises one or more of the CDRs of the anti-LRP5/6 antibodies described herein. In this regard, it has been shown in some cases that the transfer of only the VHCDR3 of an antibody can be performed while still retaining desired specific binding (Barbas et al., PNAS (1995) 92: 2529-2533). See also, McLane et al., PNAS (1995) 92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

Also disclosed herein is a method for obtaining an antibody or antigen binding domain specific for a Fzd receptor, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein or a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for one or more Fzd receptor and optionally with one or more desired properties. The VL domains may have an amino acid sequence which is substantially as set out herein. An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

In particular embodiments, Wnt surrogate molecules are water soluble. By “water soluble” it is meant a composition that is soluble in aqueous buffers in the absence of detergent, usually soluble at a concentration that provides a biologically effective dose of the polypeptide. Compositions that are water soluble form a substantially homogenous composition that has a specific activity that is at least about 5% that of the starting material from which it was purified, usually at least about 10%, 20%, or 30% that of the starting material, more usually about 40%, 50%, or 60% that of the starting material, and may be about 50%, about 90% or greater. Wnt surrogate molecules disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of at least 25 μM and higher, e.g. at least 25 μM, 40 μM, or 50 μM, usually at least 60 μM, 70 μM, 80 μM, or 90 μM, sometimes as much as 100 μM, 120 μM, or 150 μM. In other words, Wnt surrogate molecules disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of about 0.1 mg/ml, about 0.5 mg/ml, of about 1 mg/ml or more.

An antigen or epitope that “specifically binds” or “preferentially binds” (used interchangeably herein) to an antibody or antigen-binding fragment thereof is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule, e.g., a Wnt surrogate molecule, is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. A molecule or binding region thereof, e.g., a Wnt surrogate molecule or binding region thereof, “specifically binds” or “preferentially binds” to a target antigen, e.g., a Fzd receptor, if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, a Wnt surrogate molecule or binding region thereof that specifically or preferentially binds to the Fzd1 receptor is an antibody that binds to the Fzd1 receptor with greater affinity, avidity, more readily, and/or with greater duration than it binds to other Fzd receptors or non-Fzd proteins. It is also understood by reading this definition that, for example, a Wnt surrogate molecule or binding region thereof that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

In some embodiments, any of the one or more Fzd binding region of a Wnt surrogate molecule binds to one, two, three, four, five or more different frizzled proteins, e.g., one or more of human frizzled proteins Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, Fzd10. In some embodiments, any of the Fzd binding regions binds to Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8. In various embodiments, any of the Fzd binding regions binds to: (i) Fzd1, Fzd2, Fzd7 and Fzd9; (ii) Fzd1, Fzd2 and Fzd7; (iii) Fzd5 and Fzd8; (iv) Fzd5, Fzd7 and Fzd8; (v) Fzd1, Fzd4, Fzd5 and Fzd8; (vi) Fzd1, Fzd2, Fzd5, Fzd7 and Fzd8; (vii) Fzd4 and Fzd9; (viii) Fzd9 and Fzd10; (ix) Fzd5, Fzd8 and Fzd10; or (x) Fzd4, Fzd5 and Fzd8; Fzd1, Fzd5, Fzd7 and Fzd8. In some embodiments, the Fzd binding region is selective for one or more Fzd protein of interest, e.g. having a specificity for the one or more desired Fzd protein of at least 10-fold, 25-fold, 50-fold, 100-fold, 200-fold or more relative to other Fzd proteins. In some embodiments, any of the one or more Fzd binding region of a Wnt surrogate molecule is monospecific and binds or specifically binds to only one of Fzd1, Fzd2, Fzd3, Fzd4, Fzd5, Fzd6, Fzd7, Fzd8, Fzd9, or Fzd10.

In some embodiments, any of the one or more LRP5/6 binding region of a Wnt surrogate molecule binds to one or both of LRP5/6. For convenience, the term “LRP5/6” is used to refer collectively to either or both of LRP5 and/or LRP6.

Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K_(d)) of the interaction, wherein a smaller K_(d) represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (K_(on)) and the “off rate constant” (K_(off)) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K_(off)/K_(on) enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

In certain embodiments, the Wnt surrogate molecules or binding regions thereof described herein have an affinity of less than about 10,000, less than about 1000, less than about 100, less than about 10, less than about 1 or less than about 0.1 nM, and in some embodiments, the antibodies may have even higher affinity for one or more Fzd receptor or LRP5 or LRP6 receptor.

The constant regions of immunoglobulins show less sequence diversity than the variable regions, and are responsible for binding a number of natural proteins to elicit important biochemical events. In humans, there are five different classes of antibodies including IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The distinguishing features between these antibody classes are their constant regions, although subtler differences may exist in the V region.

The Fc region of an antibody interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector functions. For IgG, the Fc region comprises Ig domains CH2 and CH3 and the N-terminal hinge leading into CH2. An important family of Fc receptors for the IgG class are the Fc gamma receptors (FcγRs). These receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans this protein family includes FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a membrane spanning region, and an intracellular domain that may mediate some signaling event within the cell. These receptors are expressed in a variety of immune cells including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells. Formation of the Fc/FcγR complex recruits these effector cells to sites of bound antigen, typically resulting in signaling events within the cells and important subsequent immune responses such as release of inflammation mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.

The ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells. The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is referred to as antibody dependent cell-mediated phagocytosis (ADCP). All FcγRs bind the same region on Fc, at the N-terminal end of the Cg2 (CH2) domain and the preceding hinge. This interaction is well characterized structurally (Sondermann et al., 2001, J Mol Biol 309:737-749), and several structures of the human Fc bound to the extracellular domain of human FcγRIIIb have been solved (pdb accession code 1E4K) (Sondermann et al., 2000, Nature 406:267-273.) (pdb accession codes 11 IIS and 11 IX) (Radaev et al., 2001, J Biol Chem 276:16469-16477.)

The different IgG subclasses have different affinities for the FcγRs, with IgG1 and IgG3 typically binding substantially better to the receptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett 82:57-65). All FcγRs bind the same region on IgG Fc, yet with different affinities: the high affinity binder FcγRI has a K_(d) for IgG1 of 10⁻⁸ M⁻¹, whereas the low affinity receptors FcγRII and FcγRIII generally bind at 10⁻⁶ and 10⁻⁵ respectively. The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical; however, FcγRIIIb does not have an intracellular signaling domain. Furthermore, whereas FcγRI, FcγRIIa/c, and FcγRIIIa are positive regulators of immune complex-triggered activation, characterized by having an intracellular domain that has an immunoreceptor tyrosine-based activation motif (ITAM), FcγRIIb has an immunoreceptor tyrosine-based inhibition motif (ITIM) and is therefore inhibitory. Thus the former are referred to as activation receptors, and FcγRIIb is referred to as an inhibitory receptor. The receptors also differ in expression pattern and levels on different immune cells. Yet another level of complexity is the existence of a number of FcγR polymorphisms in the human proteome. A particularly relevant polymorphism with clinical significance is V158/F158 FcγRIIIa. Human IgG1 binds with greater affinity to the V158 allotype than to the F158 allotype. This difference in affinity, and presumably its effect on ADCC and/or ADCP, has been shown to be a significant determinant of the efficacy of the anti-CD20 antibody rituximab (Rituxan®, a registered trademark of IDEC Pharmaceuticals Corporation). Subjects with the V158 allotype respond favorably to rituximab treatment; however, subjects with the lower affinity F158 allotype respond poorly (Cartron et al., 2002, Blood 99:754-758). Approximately 10-20% of humans are V158/V158 homozygous, 45% are V158/F158 heterozygous, and 35-45% of humans are F158/F158 homozygous (Lehrbecher et al., 1999, Blood 94:4220-4232; Cartron et al., 2002, Blood 99:754-758). Thus 80-90% of humans are poor responders, that is, they have at least one allele of the F158 FcγRIIIa.

The Fc region is also involved in activation of the complement cascade. In the classical complement pathway, C1 binds with its C1q subunits to Fc fragments of IgG or IgM, which has formed a complex with antigen(s). In certain embodiments of the invention, modifications to the Fc region comprise modifications that alter (either enhance or decrease) the ability of a Fzd-specific antibody as described herein to activate the complement system (see e.g., U.S. Pat. No. 7,740,847). To assess complement activation, a complement-dependent cytotoxicity (CDC) assay may be performed (See, e.g., Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996)).

Thus in certain embodiments, the present invention provides anti-Fzd antibodies having a modified Fc region with altered functional properties, such as reduced or enhanced CDC, ADCC, or ADCP activity, or enhanced binding affinity for a specific FcγR or increased serum half-life. Other modified Fc regions contemplated herein are described, for example, in issued U.S. Pat. Nos. 7,317,091; 7,657,380; 7,662,925; 6,538,124; 6,528,624; 7,297,775; 7,364,731; Published U.S. Applications US2009092599; US20080131435; US20080138344; and published International Applications WO2006/105338; WO2004/063351; WO2006/088494; WO2007/024249.

In certain embodiments, Wnt surrogate molecules comprise antibody variable domains with the desired binding specificities fused to immunoglobulin constant domain sequences. In certain embodiments, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C_(H)2, and C_(H)3 regions. In particular embodiments, the first heavy-chain constant region (C_(H)1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.

Wnt surrogate molecules disclosed herein may also be modified to include an epitope tag or label, e.g., for use in purification or diagnostic applications. There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52: 127-131 (1992). The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.

In certain embodiments, anti-LRP5/6 antibodies and antigen-binding fragments thereof and/or anti-Fzd antibodies and antigen-binding fragments thereof present within a Wnt surrogate molecule are monoclonal. In certain embodiments, they are humanized.

The present invention further provides in certain embodiments an isolated nucleic acid encoding a polypeptide present in a Wnt surrogate molecule disclosed herein. Nucleic acids include DNA and RNA. These and related embodiments may include polynucleotides encoding antibody fragments that bind one or more Fzd receptors and/or LRP5 or LRP6 as described herein. The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature; (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. An isolated polynucleotide may include naturally occurring and/or artificial sequences.

The term “operably linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a transcription control sequence “operably linked” to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

The term “control sequence” as used herein refers to polynucleotide sequences that can affect expression, processing or intracellular localization of coding sequences to which they are ligated or operably linked. The nature of such control sequences may depend upon the host organism. In particular embodiments, transcription control sequences for prokaryotes may include a promoter, ribosomal binding site, and transcription termination sequence. In other particular embodiments, transcription control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences and polyadenylation sequences. In certain embodiments, “control sequences” can include leader sequences and/or fusion partner sequences.

The term “polynucleotide” as referred to herein means single-stranded or double-stranded nucleic acid polymers. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2′,3′-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term “polynucleotide” specifically includes single and double stranded forms of DNA.

The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” includes nucleotides with modified or substituted sugar groups and the like. The term “oligonucleotide linkages” includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for any purpose. An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.

The term “vector” is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell. The term “expression vector” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

As will be understood by those skilled in the art, polynucleotides may include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the skilled person.

As will be also recognized by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide according to the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. Polynucleotides may comprise a native sequence or may comprise a sequence that encodes a variant or derivative of such a sequence.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encodes an antibody as described herein. Some of these polynucleotides bear minimal sequence identity to the nucleotide sequence of the native or original polynucleotide sequence encoding a polypeptide within a Wnt surrogate molecule. Nonetheless, polynucleotides that vary due to differences in codon usage are expressly contemplated by the present disclosure. In certain embodiments, sequences that have been codon-optimized for mammalian expression are specifically contemplated.

Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, may be employed for the preparation of variants and/or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provide a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

In certain embodiments, the inventors contemplate the mutagenesis of the polynucleotide sequences that encode a polypeptide present in a Wnt surrogate molecule, to alter one or more properties of the encoded polypeptide, such as the binding affinity, or the function of a particular Fc region, or the affinity of the Fc region for a particular FcγR. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phages are readily commercially available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.

The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

In many embodiments, one or more nucleic acids encoding a polypeptide of a Wnt surrogate molecule are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded polypeptides. The Wnt surrogate polypeptides of this disclosure may be prepared using standard techniques well known to those of skill in the art in combination with the polypeptide and nucleic acid sequences provided herein. The polypeptide sequences may be used to determine appropriate nucleic acid sequences encoding the particular polypeptide disclosed thereby. The nucleic acid sequence may be optimized to reflect particular codon “preferences” for various expression systems according to standard methods well known to those of skill in the art.

According to certain related embodiments there is provided a recombinant host cell which comprises one or more constructs as described herein, e.g., a vector comprising a nucleic acid encoding a Wnt surrogate molecule or polypeptide thereof; and a method of production of the encoded product, which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, an antibody or antigen-binding fragment thereof, may be isolated and/or purified using any suitable technique, and then used as desired.

Polypeptides, and encoding nucleic acid molecules and vectors, may be isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the desired function. Nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli.

The expression of polypeptides, e.g., antibodies and antigen-binding fragments thereof, in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates thereto.

The term “host cell” is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the herein described polypeptides, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any herein described polypeptide. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present. Accordingly there is also contemplated a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. In one embodiment, the nucleic acid is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance-with standard techniques.

The present invention also provides, in certain embodiments, a method which comprises using a construct as stated above in an expression system in order to express a particular polypeptide such as a Wnyt mimetic molecule as described herein. The term “transduction” is used to refer to the transfer of genes from one bacterium to another, usually by a phage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses. The term “transfection” is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA. For example, a cell is transformed where it is genetically modified from its native state. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated with the division of the cell. The term “naturally occurring” or “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by a human. Similarly, “non-naturally occurring” or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by a human.

The terms “polypeptide” “protein” and “peptide” and “glycoprotein” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms “polypeptide” or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms “polypeptide” and “protein” specifically encompass Wnt surrogate molecules, Fzd binding regions thereof, LRP5/6 binding regions thereof, antibodies and antigen-binding fragments thereof that bind to a Fzd receptor or a LRP5 or LRP6 receptor disclosed herein, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of any of these polypeptides. Thus, a “polypeptide” or a “protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains.

The term “isolated protein,” “isolated Wnt surrogate molecule or “isolated antibody” referred to herein means that a subject protein, Wnt surrogate molecule, or antibody: (1) is free of at least some other proteins with which it would typically be found in nature; (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species; (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature; (5) is not associated (by covalent or noncovalent interaction) with portions of a protein with which the “isolated protein” is associated in nature; (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature; or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein may comprise naturally-occurring and/or artificial polypeptide sequences. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

Amino acid sequence modification(s) of any of the polypeptides (e.g., Wnt surrogate molecules or Fzd binding regions or LRP5/6 binding regions thereof) described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the Wnt surrogate molecule. For example, amino acid sequence variants of a Wnt surrogate molecule may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final Wnt surrogate molecule, provided that the final construct possesses the desired characteristics (e.g., high affinity binding to one or more Fzd and/or LRP5/6 receptor). The amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.

The present disclosure provides variants of any of the polypeptides (e.g., Wnt surrogate molecules or Fzd binding regions or LRP5/6 binding regions thereof, or antibodies or antigen-binding fragments thereof) disclosed herein. In certain embodiments, a variant has at least 90%, at least 95%, at least 98%, or at least 99% identity to a polypeptide disclosed herein. In certain embodiments, such variant polypeptides bind to one or more Fzd receptor, and/or to one or more LRP5/6 receptor, at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a Wnt surrogate molecule specifically set forth herein. In further embodiments, such variant Wnt surrogate molecules bind to one or more Fzd receptor, and/or to one or more LRP5/6 receptor, with greater affinity than the Wnt surrogate molecules set forth herein, for example, that bind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% as well as an antibody sequence specifically set forth herein.

In particular embodiments, the Wnt surrogate molecule or a binding region thereof, e.g., a Fab, scFv, or VHH or sdAb may comprise: a) a heavy chain variable region comprising: i. a CDR1 region that is identical in amino acid sequence to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the selected antibody; and/or b) a light chain variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the light chain CDR1 region of the selected antibody; ii. a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the selected antibody; wherein the antibody specifically binds a selected target (e.g., one or more Fzd receptors or LRP5 or LRP6 receptors). In a further embodiment, the antibody, or antigen-binding fragment thereof, is a variant antibody or antigen-binding fragment thereof wherein the variant comprises a heavy and light chain identical to the selected antibody except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in the CDR regions of the VH and VL regions. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15 more amino acid substitutions in the CDR regions of the selected antibody. Substitutions may be in CDRs either in the VH and/or the VL regions. (See e.g., Muller, 1998, Structure 6:1153-1167).

In particular embodiments, the Wnt surrogate molecule or a binding region thereof, e.g., a Fab, scFv, or VHH or sdAb may have: a) a heavy chain variable region having an amino acid sequence that is at least 80% identical, at least 95% identical, at least 90%, at least 95% or at least 98% or 99% identical, to the heavy chain variable region of an antibody or antigen-binding fragments thereof described herein; and/or b) a light chain variable region having an amino acid sequence that is at least 80% identical, at least 85%, at least 90%, at least 95% or at least 98% or 99% identical, to the light chain variable region of an antibody or antigen-binding fragments thereof described herein. The amino acid sequence of illustrative antigen-binding fragments thereof are set forth in SEQ ID NOs:1-128.

A polypeptide has a certain percent “sequence identity” to another polypeptide, meaning that, when aligned, that percentage of amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)

Of interest is the BestFit program using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.

Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters:

Mismatch Penalty: 1.00; Gap Penalty: 1.00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.

In particular embodiments, the Wnt surrogate molecule or a binding region thereof, e.g., a Fab, scFv, or VHH or sdAb may comprise: a) a heavy chain variable region comprising: i. a CDR1 region that is identical in amino acid sequence to the heavy chain CDR1 region of a selected antibody described herein; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the selected antibody; and b) a light chain variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the light chain CDR1 region of the selected antibody; ii. a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the selected antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the selected antibody; wherein the antibody specifically binds a selected target (e.g., a Fzd receptor, such as Fzd1). In a further embodiment, the antibody, or antigen-binding fragment thereof, is a variant antibody wherein the variant comprises a heavy and light chain identical to the selected antibody except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions in the CDR regions of the VH and VL regions. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments, 9, 10, 11, 12, 13, 14, 15 more amino acid substitutions in the CDR regions of the selected antibody. Substitutions may be in CDRs either in the VH and/or the VL regions. (See e.g., Muller, 1998, Structure 6:1153-1167).

Determination of the three-dimensional structures of representative polypeptides (e.g., variant Fzd binding regions or LRP5/6 binding regions of Wnt surrogate molecules as provided herein) may be made through routine methodologies such that substitution, addition, deletion or insertion of one or more amino acids with selected natural or non-natural amino acids can be virtually modeled for purposes of determining whether a so derived structural variant retains the space-filling properties of presently disclosed species. See, for instance, Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244 (2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature 450:259 (2007); Raman et al. Science 327:1014-1018 (2010). Some additional non-limiting examples of computer algorithms that may be used for these and related embodiments, such as for rational design of binding regions include VMD which is a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting (see the website for the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne, at ks.uiuc.edu/Research/vmd/. Many other computer programs are known in the art and available to the skilled person and which allow for determining atomic dimensions from space-filling models (van der Waals radii) of energy-minimized conformations; GRID, which seeks to determine regions of high affinity for different chemical groups, thereby enhancing binding, Monte Carlo searches, which calculate mathematical alignment, and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106: 765), which assess force field calculations, and analysis (see also, Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. Health Perspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488). A variety of appropriate computational computer programs are also commercially available, such as from Schrödinger (Munich, Germany).

Compositions

Pharmaceutical compositions comprising a Wnt surrogate molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed. In particular embodiments, the pharmaceutical composition further comprises one or more Wnt polypeptides or Norrin polypeptides.

In further embodiments, pharmaceutical compositions comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt surrogate molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed. In particular embodiments, the pharmaceutical composition further comprises one or more polynucleotides comprising a nucleic acid sequence encoding a Wnt polypeptide or Norrin polypeptide. In certain embodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotides are modified mRNAs further comprising a 5′ cap sequence and/or a 3′ tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same polynucleotide.

In further embodiments, pharmaceutical compositions comprising an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt surrogate molecule described herein and one or more pharmaceutically acceptable diluent, carrier, or excipient are also disclosed. In particular embodiments, the pharmaceutical composition further comprises an expression vector, e.g., a viral vector, comprising a polynucleotide comprising a nucleic acid sequence encoding a Wnt polypeptide or Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., expression cassette.

The present invention further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operatively linked to a nucleic acid encoding a Wnt surrogate molecule and one or more pharmaceutically acceptable diluent, carrier, or excipient. In particular embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operatively linked to a nucleic acid sequence encoding a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same polynucleotide, e.g., expression cassette and/or in the same cell. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In particular embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

The present disclosure contemplates pharmaceutical compositions comprising a first molecule for delivery of a Wnt surrogate molecule as a first active agent and a second molecule for delivery of a Wnt polypeptide or Norrin polypeptide. The first and second molecule may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecule may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids encoding the first or second active agent (optionally DNA or mRNA, optionally modified RNA), vectors comprising a nucleic acid sequence encoding the first or second active agent (optionally expression vectors or viral vectors), and cells comprising a nucleic acid sequence encoding the first or second active agent (optionally an expression cassette).

The subject molecules, alone or in combination, can be combined with pharmaceutically-acceptable carriers, diluents, excipients and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile.

Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition is sterile and should be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the anti-Fzd antibody or antigen-binding fragment thereof (or encoding polynucleotide or cell comprising the same) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the pharmaceutical compositions are prepared with carriers that will protect the antibody or antigen-binding fragment thereof against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

It may be advantageous to formulate the pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the antibody or antigen-binding fragment thereof and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active antibody or antigen-binding fragment thereof for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser, e.g. syringe, e.g. a prefilled syringe, together with instructions for administration.

The pharmaceutical compositions of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal comprising a human, is capable of providing (directly or indirectly) the biologically active antibody or antigen-binding fragment thereof.

The present invention includes pharmaceutically acceptable salts of a Wnt surrogate molecule described herein. The term “pharmaceutically acceptable salt” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. A variety of pharmaceutically acceptable salts are known in the art and described, e.g., in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). Also, for a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Metals used as cations comprise sodium, potassium, magnesium, calcium, and the like. Amines comprise N—N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.

In some embodiments, the pharmaceutical composition provided herein comprise a therapeutically effective amount of a Wnt surrogate molecule or pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable carrier, diluent and/or excipient, for example saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars and the like are octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, camitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, this formulation is stable for at least six months at 4° C.

In some embodiments, the pharmaceutical composition provided herein comprises a buffer, such as phosphate buffered saline (PBS) or sodium phosphate/sodium sulfate, tris buffer, glycine buffer, sterile water and other buffers known to the ordinarily skilled artisan such as those described by Good et al. (1966) Biochemistry 5:467. The pH of the buffer may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

Methods of Use

The present disclosure also provides methods for using the Wnt surrogate molecules disclosed herein, e.g., to modulate a Wnt signaling pathway, e.g., to increase Wnt signaling, and the administration of a Wnt surrogate molecule disclosed herein in a variety of therapeutic settings. Provided herein are methods of treatment using a Wnt surrogate molecule. In one embodiment, a Wnt surrogate molecule is provided to a subject having a disease involving inappropriate or deregulated Wnt signaling, e.g., reduced Wnt signaling.

Increasing Wnt Pathway Signaling and Related Therapeutic Methods

In certain embodiments, a Wnt surrogate molecule may be used to increase Wnt signaling in a tissue or cell. Thus, in some aspects, the present invention provides a method for increasing Wnt signaling or enhancing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a Wnt surrogate molecule or pharmaceutically acceptable salt thereof disclosed herein, wherein the a Wnt surrogate molecule is a Wnt signaling pathway agonist. In some embodiments, contacting occurs in vitro, ex vivo, or in vivo. In particular embodiments, the cell is a cultured cell, and the contacting occurs in vitro. In certain embodiments, the method comprises further contacting the tissue or cell with one or more Wnt polypeptides or Norrin polypeptides.

In related aspects, the present invention provides a method for increasing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a polynucleotide comprising a Wnt surrogate molecule disclosed herein. In certain embodiments, the target tissue or cell is also contacted with a polynucleotide comprising a nucleic acid sequence that encodes a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotides are modified mRNAs further comprising a 5′ cap sequence and/or a 3′ tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same polynucleotide.

In related aspects, the present invention provides a method for increasing Wnt signaling in a tissue or cell, comprising contacting the tissue or cell with an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt surrogate molecule. In certain embodiments, the tissue or cell is also contacted with a vector comprising a nucleic acid sequence that encodes a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector, and may comprise a promoter operatively linked to the nucleic acid sequence. In particular embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding a Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same vector, e.g., in the same expression cassette.

In related aspects, the present invention provides a method for increasing Wnt signaling in a tissue, comprising contacting the tissue with an effective amount of a cell comprising a nucleic acid sequence encoding a Wnt surrogate molecule of the present invention. In certain embodiments, the tissue is also contacted with a cell comprising a nucleic acid sequence that encodes a Wnt polypeptide or Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same cell. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell was transduced with a vector comprising an expression cassette encoding the Wnt surrogate molecule or the Wnt polypeptide or Norrin polypeptide. In particular embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

Wnt surrogate molecules disclosed herein may be used in to treat a disease, disorder or condition, for example, by increasing Wnt signaling in a targeted cell, tissue or organ. Thus, in some aspects, the present invention provides a method for treating a disease or condition in a subject in need thereof, e.g., a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting the subject with an effective amount of a composition of the present disclosure. In particular embodiments, the composition is a pharmaceutical composition comprising any of: a Wnt surrogate molecule; a polynucleotide comprising a nucleic acid sequence encoding a Wnt surrogate molecule, e.g., a DNA or mRNA, optionally a modified mRNA; a vector comprising a nucleic acid sequence encoding a Wnt surrogate molecule, e.g., an expression vector or viral vector; or a cell comprising a nucleic acid sequence encoding a Wnt surrogate molecule, e.g., a cell transduced with an expression vector or viral vector encoding a Wnt surrogate molecule. In particular embodiments, the disease or condition is a pathological disease or disorder, or an injury, e.g., an injury resulting from a wound. In certain embodiments, the wound may be the result of another therapeutic treatment. In certain embodiments, the disease or condition comprises impaired tissue repair, healing or regeneration, or would benefit from increased tissue repair, healing or regeneration. In some embodiments, contacting occurs in vivo, i.e., the subject composition is administered to a subject.

In certain embodiments, the method comprises further contacting the subject with a pharmaceutical composition comprising one or more Wnt polypeptides or Norrin polypeptides. The present disclosure contemplates contacting a subject with a first molecule for delivery of a Wnt surrogate molecule as a first active agent and a second molecule for delivery of a Wnt polypeptide or Norrin polypeptide. The first and second molecule may be the same type of molecule or different types of molecules. For example, in certain embodiments, the first and second molecule may each be independently selected from the following types of molecules: polypeptides, small organic molecules, nucleic acids encoding the first or second active agent (optionally DNA or mRNA, optionally modified RNA), vectors comprising a nucleic acid sequence encoding the first or second active agent (optionally expression vectors or viral vectors), and cells comprising a nucleic acid sequence encoding the first or second active agent (optionally an expression cassette).

In related aspects, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence encoding a Wnt surrogate molecule disclosed herein. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a polynucleotide comprising a nucleic acid sequence that encodes a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the polynucleotides are DNA or mRNA, e.g., a modified mRNA. In particular embodiments, the polynucleotides are modified mRNAs further comprising a 5′ cap sequence and/or a 3′ tailing sequence, e.g., a polyA tail. In other embodiments, the polynucleotides are expression cassettes comprising a promoter operatively linked to the coding sequences. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same polynucleotide.

In related aspects, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence encoding a Wnt surrogate molecule. In certain embodiments, the subject is also contacted with a pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid sequence that encodes a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the vector is an expression vector, and may comprise a promoter operatively linked to the nucleic acid sequence. In particular embodiments, the vector is a viral vector. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same vector, e.g., in the same expression cassette.

In related aspects, the present invention provides a method for treating a disease or condition, e.g., a disease or disorder associated with reduced Wnt signaling, or for which increased Wnt signaling would provide a therapeutic benefit, comprising contacting a subject in need thereof with a pharmaceutical composition comprising an effective amount of a cell comprising a nucleic acid sequence encoding a Wnt surrogate molecule. In certain embodiments, the subject is also contacted with a cell comprising a nucleic acid sequence that encodes a Wnt polypeptide or a Norrin polypeptide. In certain embodiments, the nucleic acid sequence encoding the Wnt surrogate molecule and the nucleic acid sequence encoding the Wnt polypeptide or Norrin polypeptide are present in the same cell. In particular embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated. In certain embodiments, the cell was transduced with a vector comprising an expression cassette encoding the Wnt surrogate molecule or the Wnt polypeptide or Norrin polypeptide. In particular embodiments, the cell is a stem cell, e.g., an adipose-derived stem cell or a hematopoietic stem cell.

Wnt signaling plays key roles in the developmental process and maintenance of stem cells. Reactivation of Wnt signals is associated with regeneration and repair of most tissues after injuries and diseases. Wnt surrogate molecule molecules are expected to provide benefit of healing and tissue repair in response to injuries and diseases. Causes of tissue damage and loss include but are not limited to aging, degeneration, hereditary conditions, infection and inflammation, traumatic injuries, toxins/metabolic-induced toxicities, or other pathological conditions. Wnt signals and enhancers of Wnt signals have been shown to activate adult, tissue-resident stem cells. In some embodiments, the compounds of the invention are administered for use in treating diseased or damaged tissue, for use in tissue regeneration and for use in cell growth and proliferation, and/or for use in tissue engineering.

Human diseases associated with mutations of the Wnt pathway provide strong evidence for enhancement of Wnt signals in the treatment and prevention of diseases. Preclinical in vivo and in vitro studies provide additional evidence of involvement of Wnt signals in many disease conditions and further support utilization of a Wnt surrogate molecule in various human diseases. For example, compositions of the present invention may be used to promote or increase bone growth or regeneration, bone grafting, healing of bone fractures, treatment of osteoporosis and osteoporotic fractures, spinal fusion, spinal cord injuries, including vertebral compression fractures, pre-operative spinal surgery optimization, osseointegration of orthopedic devices, tendon-bone integration, tooth growth and regeneration, dental implantation, periodontal diseases, maxillofacial reconstruction, and osteonecrosis of the jaw. They may also be used in the treatment of alopecia; enhancing regeneration of sensory organs, e.g. treatment of hearing loss, including regeneration of inner and outer auditory hair cells treatment of vestibular hypofunction, treatment of macular degeneration, treatment of retinopathies, including vitreoretinopathy, diabetic retinopathy, other diseases of retinal degeneration, Fuchs' dystrophy, other cornea disease, etc.; treatment of stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis, multiple dystrophy, muscle atrophy as a result of sarcopenia or cachexia, and other conditions affecting the degeneration or integrity of the blood brain barrier; The compositions of this invention may also be used in treatment of oral mucositis, treatment of short bowel syndrome, inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), in particular CD with fistula formation, other gastrointestinal disorders; treatment of metabolic syndrome, dyslipidemia, treatment of diabetes, treatment of pancreatitis, conditions where exocrine or endocrine pancreas tissues are damaged; conditions where enhanced epidermal regeneration is desired, e.g., epidermal wound healing, treatment of diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, etc., conditions where angiogenesis is beneficial; treatment of myocardial infarction, coronary artery disease, heart failure; enhanced growth of hematopoietic cells, e.g. enhancement of hematopoietic stem cell transplants from bone marrow, mobilized peripheral blood, treatment of immunodeficiencies, graft versus host diseases, etc.; treatment of acute kidney injuries, chronic kidney diseases; treatment of lung diseases, chronic obstructive pulmonary diseases (COPD), pulmonary fibrosis, including idiopathic pulmonary fibrosis, enhanced regeneration of lung tissues. The compositions of the present invention may also be used in enhanced regeneration of liver cells, e.g. liver regeneration, treatment of cirrhosis, enhancement of liver transplantations, treatment of acute liver failure, treatment of chronic liver diseases with hepatitis C or B virus infection or post-antiviral drug therapies, alcoholic liver diseases, including alcoholic hepatitis, non-alcoholic liver diseases with steatosis or steatohepatitis, and the like. The compositions of this invention may treat diseases and disorders including, without limitation, conditions in which regenerative cell growth is desired.

Human genetics involving loss-of-function or gain-of-function mutations in Wnt signaling components show strong evidence supporting enhancing Wnt signals for bone growth. Conditions in which enhanced bone growth is desired may include, without limitation, fractures, grafts, ingrowth around prosthetic devices, osteoporosis, osteoporotic fractures, spinal fusion, vertebral compression fractures, pre-operative optimization for spinal surgeries, osteonecrosis of the jaw, dental implantation, periodontal diseases, maxillofacial reconstruction, and the like. Wnt surrogate molecules enhance and promotes Wnt signals which are critical in promoting bone regeneration. Methods for regeneration of bone tissues benefit from administration of the compounds of the invention, which can be systemic or localized. In some embodiments, bone marrow cells are exposed to molecules of the invention, such that stem cells within that marrow become activated.

In some embodiments, bone regeneration is enhanced by contacting a responsive cell population, e.g. bone marrow, bone progenitor cells, bone stem cells, etc. with an effective dose of a Wnt surrogate molecule disclosed herein. Methods for regeneration of bone tissues benefit from administration of the Wnt surrogate molecule which can be systemic or localized. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, polymeric microspheres, nanoparticles, bone cements, and the like.

In particular embodiments, compositions comprising one or more Wnt surrogate molecule disclosed herein (or a polynucleotide encoding a Wnt surrogate molecule, or a vector or cell comprising a polynucleotide encoding a Wnt surrogate molecule) are used to treat or prevent a bone disease or disorder, including but not limited to any of the following, or to treat or prevent an injury associated with, but not limited to, any of the following: osteoporosis, osteoporotic fractures, bone fractures including vertebral compression fractures, non-union fractures, delayed union fractures, spinal fusion, osteonecrosis, osteonecrosis of the jaw, hip, femoral head, etc., osseointegration of implants (e.g., to accelerate recovery following partial or total knee or hip replacement), osteogenesis imperfecta, bone grafts, tendon repair, maxillofacial surgery, dental implant, all other bone disorders or defects resulting from genetic diseases, degeneration, aging, drugs, or injuries. In one embodiment, Wnt surrogate molecules that bind Fzd1, Fzd 2, and Fzd 7, and also LRP5 and/or LRP6, are used to treat or prevent any bone disease or disorder. In one embodiment, Wnt surrogate molecules that bind Fzd1, Fzd 2, Fzd 5, Fzd 7 and Fzd 8, and also LRP5 and/or LRP6, are used to treat or prevent any bone disease or disorder. Other Fzd molecules that bind to additional Fzd receptors can also be used with LRP5 and/or LRP6 binders.

In particular embodiments, compositions and methods disclosed herein may be used to: increase bone mineral density, increase bone volume (e.g., tibia and/or femur bone volume), increase cortical thickness (e.g., in trabecular region or in femur mid-diaphysis), increase mineral apposition rate, increase the number of osteblasts and/or decrease the number of osteoclasts (e.g., in bone), increase bone stiffness, increase the ultimate load to fracture point, improve bone resistance to fracture, decrease bone resorption, decrease bone loss associated with osteoporosis, or increase biochemical strength of bone, in a subject. In one embodiment, Wnt surrogate molecules that bind Fzd1, Fzd 2, and Fzd 7 are used for any of these indicated uses. In one embodiment, Wnt surrogate molecules that bind Fzd1, Fzd 2, Fzd 5, Fzd 7 and Fzd 8 are used for any of these indicated uses.

Methods disclosed herein, including methods for treating or preventing a bone disease or disorder include methods that comprise providing to a subject in need thereof both a Wnt surrogate molecule and an antiresorptive agent. In certain embodiments, the methods are used for the treatment of osteoporosis, optionally post-menopausal osteoporosis.

The disclosure also provides a method for inhibiting or reducing bone resorption in a subject in need thereof, comprising providing to the subject an effective amount of a Wnt surrogate molecule, wherein the Wnt surrogate molecule is an agonist of a Wnt signaling pathway. In certain embodiments, the method further comprises providing to the subject an antiresorptive agent. In certain embodiments, the subject has been diagnosed with or is at risk for osteoporosis, optionally postmenopausal osteoporosis. A variety of antiresorptive agents are known in the art and include, but are not limited to, those disclosed herein.

When a Wnt surrogate molecule is provide to the subject in combination with another therapeutic agent, such as an antiresorptive agent, the two agent may be provided in the same or different pharmaceutical compositions. They may be provided to the subject at the same time, at different times, e.g., simultaneously, consecutively, or during overlapping or non-overlapping time periods. In certain embodiments, the two agents are therapeutically active in the subject during an overlapping time period.

Compositions comprising one or more Wnt surrogate molecule disclosed herein (or a polynucleotide encoding a Wnt surrogate molecule, or a vector or cell comprising a polynucleotide encoding a Wnt surrogate molecule) can be used for the in vivo treatment of skeletal tissue deficiencies. By “skeletal tissue deficiency”, it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions. The compositions of the present invention can be used as part of a regimen for restoring cartilage function to a connective tissue, for the repair of defects or lesions in cartilage tissue such as degenerative wear and arthritis, trauma to the tissue, displacement of torn meniscus, meniscectomy, a luxation of a joint by a torn ligament, malalignment of joints, bone fracture, or by hereditary disease.

A Wnt surrogate molecule may also be used for treatment of periodontal diseases. Periodontal diseases are a leading cause of tooth loss and are linked to multiple systemic conditions. In some embodiments, tooth or underlying bone regeneration is enhanced by contacting a responsive cell population. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo, with subsequent implantation of the activated stem or progenitor cells. The molecule may be localized to the site of action, e.g. by loading onto a matrix, which is optionally biodegradable, and optionally provides for a sustained release of the active agent. Matrix carriers include, without limitation, absorbable collagen sponges, ceramics, hydrogels, bone cements, polymeric microspheres, nanoparticles, and the like.

Studies have shown that biology of Wnt signaling and R-spondins are capable of promoting sensory hair cell regeneration in the inner ear following injuries, aging, or degeneration. Loss of sensory hair cells in the inner ear involved in hearing loss or vestibular hypofunction may also benefit from the compositions of the invention. In the inner ear, the auditory organ houses mechanosensitive hair cells required for translating sound vibration to electric impulses. The vestibular organs, comprised of the semicircular canals (SSCs), the utricle, and the saccule, also contain sensory hair cells in order to detect head position and motion. Compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the ear for enhancement of auditory regeneration.

A Wnt surrogate molecule may also be used in regeneration of retinal tissue. In the adult mammalian retina, Muller glia cells are capable of regenerating retinal cells, including photoreceptors, for example after neurotoxic injury in vivo. Wnt signaling and enhancers of Wnt signals can promote proliferation of Muller glia-derived retinal progenitors after damage or during degeneration. The compositions of the invention may also be used in the regeneration of tissues and other cell types in the eye. For examples age-related macular degeneration (AMD), other retina degenerative diseases, cornea diseases, Fuchs' dystrophy, vitreoretinopathy, hereditary diseases, etc. can benefit from the compositions of the present inventions. AMD is characterized by progressively decreased central vision and visual acuity. Fuchs' dystrophy is characterized by progressive loss of cornea endothelial cells. Wnt signal and enhancing of Wnt signal can promote regeneration of cornea endothelium, retina epithelium, etc. in the eye tissue. In other embodiments, compositions of the present invention can be used, for example, in an infusion; in a matrix or other depot system; or other topical application to the eye for retinal regeneration and treatment of macular degeneration.

Specific populations of proliferating cells for homeostatic renewal of hepatocytes have been identified through lineage tracing studies, for example Axin2-positive cells in peri-central region. Lineage tracing studies also identified additional potential liver progenitor cells, including but not limited to Lgr-positive cells. The self-renewing liver cells and other populations of potential progenitor cells, including Lgr5-positive and Axin2-positive cells, are identified to be capable of regeneration responding to Wnt signals and/or R-spondins following injuries. Numerous preclinical models of acute liver injury and failure and chronic liver diseases showed recovery and regeneration of hepatocytes benefit from enhancing Wnt signals.

In certain embodiments, compositions comprising a Wnt surrogate molecule disclosed herein (or a polynucleotide encoding a Wnt surrogate molecule, or a vector or cell comprising a polynucleotide encoding a Wnt surrogate molecule) are used to promote liver regeneration, reduce fibrosis, and/or improve liver function. In certain embodiments, compositions and methods disclosed herein are used to: increase liver weight, increase the liver to body weight ratio, increase the number of PCNA and pH3 positive nuclei in liver, increase expression of Ki67 and/or Cyclin D1 in liver, increase liver cell proliferation and/or mitosis, decrease fibrosis following chronic liver injury, or increase hepatocyte function.

In particular embodiments, the compositions of this invention may be used in treatment of acute liver failure, acute alcoholic liver injuries, treatment of chronic liver diseases with hepatitis C or B virus infection or post-antiviral drug therapies, chronic alcoholic liver diseases, alcoholic hepatitis, non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis (NASH), treatment of cirrhosis and severe chronic liver diseases of all causes, and enhanced regeneration of liver cells. Methods for regeneration of liver tissue benefit from administration of the compounds of the invention, which can be systemic or localized. These include, but are not limited to, methods of systemic administration and methods of localized administration e.g. by injection into the liver tissue, by injection into veins or blood vessels leading into the liver, by implantation of a sustained release formulation, and the like.

In particular embodiments, compositions comprising a Wnt surrogate molecule disclosed herein (or a polynucleotide encoding a Wnt surrogate molecule, or a vector or cell comprising a polynucleotide encoding a Wnt surrogate molecule) are used to treat or prevent a liver disease or disorder, including but not limited to, or to treat or prevent a liver injury or disorder resulting from any of the following: acute liver failure (all causes), chronic liver failure (all causes), cirrhosis, liver fibrosis (all causes), portal hypertension, alcoholic liver diseases including alcoholic hepatitis, nonalcoholic steatohepatisis (NASH), nonalcoholic fatty liver disease (NAFLD) (fatty liver), alcoholic hepatitis, hepatitis C virus-induced liver diseases (HCV), hepatitis B virus-induced liver diseases (HBV), other viral hepatitis (e.g., hepatitis A virus-induced liver diseases (HAV) and hepatitis D virus-induced liver diseases (HDV)), primary biliary cirrhosis, autoimmune hepatitis, livery surgery, liver injury, liver transplantation, “small for size” syndrome in liver surgery and transplantation, congenital liver disease and disorders, any other liver disorder or detect resulting from genetic diseases, degeneration, aging, drugs, or injuries.

Wnt signals play an important role in regeneration of various epithelial tissues. Various epidermal conditions benefit from treatment with the compounds of the present invention. Mucositis occurs when there is a breakdown of the rapidly divided epithelial cells lining the gastro-intestinal tract, leaving the mucosal tissue open to ulceration and infection. The part of the epithelial lining that covers the mouth, called the oral mucosa, is one of the most sensitive parts of the body and is particularly vulnerable to chemotherapy and radiation. Oral mucositis is probably the most common, debilitating complication of cancer treatments, particularly chemotherapy and radiation. In addition, the compositions of the invention may also benefit treatment of short bowel syndrome, inflammatory bowel diseases (IBD), or other gastrointestinal disorders. Other epidermal conditions include epidermal wound healing, diabetic foot ulcers, syndromes involving tooth, nail, or dermal hypoplasia, and the like. Molecules of the present invention may be used in all these conditions, where regenerative cells are contacted with compounds of the invention. Methods for regeneration of epithelial tissues benefit from administration of the compounds of the invention, which can be systemic or localized. Contacting can be, for example, topical, including intradermal, subdermal, in a gel, lotion, cream etc. applied at targeted site, etc.

In addition to skin and gastrointestinal tract, Wnt signals and enhancement and promotion of Wnt signals also play an important role in repair and regeneration of tissues including pancreas, kidney, and lung in preclinical models. A Wnt surrogate molecule may benefit various disease conditions involving exocrine and endocrine pancreas, kidney, or lung. The Wnt surrogate molecules may be used in treatment of metabolic syndrome; treatment of diabetes, treatment of acute or chronic pancreatitis, exocrine pancreatic insufficiency, treatment of acute kidney injuries, chronic kidney diseases, treatment of lung diseases, including but not limited to chronic obstructive pulmonary diseases (COPD), pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), and other conditions that cause loss of lung epithelial tissues. Methods for regeneration of these tissues benefit from administration of the compounds of the invention, which can be systemic or localized.

Epidermal Wnt signaling, in coordination with signaling via other development factors, is critical for adult hair follicle regeneration. Hair loss is a common problem, and androgenetic alopecia, often called male pattern baldness, is the most common form of hair loss in men. In some embodiments, hair follicle regeneration is enhanced by contacting a responsive cell population with a molecule of the present invention. In some such embodiments, the contacting is performed in vivo. In other such embodiments, the contacting is performed ex vivo. The molecule may be localized to the site of action, e.g. topical lotions, gels, creams and the like.

Stroke, traumatic brain injury, Alzheimer's disease, multiple sclerosis and other conditions affecting the blood brain barrier (BBB) may be treated with a Wnt surrogate molecule. Angiogenesis is critical to ensure the supply of oxygen and nutrients to many tissues throughout the body, and is especially important for the CNS as the neural tissue is extremely sensitive to hypoxia and ischemia. CNS endothelial cells which form the BBB differ from endothelial cells in non-neural tissue, in that they are highly polarized cells held together by tight junctions and express specific transporters. Wnt signaling regulates CNS vessel formation and/or function. Conditions in which the BBB is compromised can benefit from administration of the compounds of the invention, which can be systemic or localized e.g. by direct injection, intrathecal administration, implantation of sustained release formulations, and the like. In addition, Wnt signal is actively involved in neurogenesis and plays a role of neuroprotection following injury. The compositions of the present invention may also be used in treatment of spinal cord injuries, other spinal cord diseases, stroke, traumatic brain injuries, etc.

Wnt signals also play a role in angiogenesis. A Wnt surrogate molecule may benefit conditions where angiogenesis is beneficial, treatment of myocardial infarction, coronary artery disease, heart failure, diabetic retinopathy, etc., and conditions from hereditary diseases. Methods for regeneration of these tissues benefit from administration of the compounds of the invention, which can be systemic or localized.

In certain embodiments, methods of the present invention promote tissue regeneration, e.g., in a tissue subjected to damage or tissue or cell reduction or loss. The loss or damage can be anything which causes the cell number to diminish, including diseases or injuries. For example, an accident, an autoimmune disorder, a therapeutic side-effect or a disease state could constitute trauma. Tissue regeneration increases the cell number within the tissue and preferably enables connections between cells of the tissue to be re-established, and more preferably the functionality of the tissue to be regained.

The terms “administering” or “introducing” or “providing”, as used herein, refer to delivery of a composition to a cell, to cells, tissues and/or organs of a subject, or to a subject. Such administering or introducing may take place in vivo, in vitro or ex vivo.

In particular embodiments, a pharmaceutical composition is administered parenterally, e.g., intravenously, orally, rectally, or by injection. In some embodiments, it is administered locally, e.g., topically or intramuscularly. In some embodiments, a composition is administered to target tissues, e.g., to bone, joints, ear tissue, eye tissue, gastrointestinal tract, skin, a wound site or spinal cord. Methods of the invention may be practiced in vivo or ex vivo. In some embodiments, the contacting of a target cell or tissue with a Wnt surrogate molecule is performed ex vivo, with subsequent implantation of the cells or tissues, e.g., activated stem or progenitor cells, into the subject. The skilled artisan can determine an appropriate site of and route of administration based on the disease or disorder being treated.

The dose and dosage regimen may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or disorder, the characteristics of the subject, and the subject's history. In particular embodiments, the amount of a Wnt surrogate molecule administered or provided to the subject is in the range of about 0.01 mg/kg to about 50 mg/kg, 0.1 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 50 mg/kg of the subject's body weight.

The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g. reducing the likelihood that the disease or symptom thereof occurs in the subject, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent (e.g., a Wnt surrogate molecule) may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. In some embodiments, the subject method results in a therapeutic benefit, e.g., preventing the development of a disorder, halting the progression of a disorder, reversing the progression of a disorder, etc. In some embodiments, the subject method comprises the step of detecting that a therapeutic benefit has been achieved. The ordinarily skilled artisan will appreciate that such measures of therapeutic efficacy will be applicable to the particular disease being modified, and will recognize the appropriate detection methods to use to measure therapeutic efficacy.

Other embodiments relate, in part, to the use of the Wnt surrogate molecules disclosed herein to promote or enhance the growth or proliferation of cells, tissues and organoids, for example, by contacting cells or tissue with one or more Wnt surrogate, optionally in combination with a Norrin or Rspondin polypeptide. In certain embodiments, the cells or tissue are contacted ex vivo, in vitro, or in vivo. Such methods may be used to generate cells, tissue or organoids for therapeutic use, e.g., to be transplanted or grafted into a subject. They may also be used to generate cells, tissue or organoids for research use. The Wnt surrogate molecules have widespread applications in non-therapeutic methods, for example in vitro research methods.

The invention provides a method for tissue regeneration of damaged tissue, such as the tissues discussed above, comprising administering a Wnt surrogate molecule to cells. The Wnt surrogate molecule may be administered directly to the cells in vivo, administered to a subject orally, intravenously, or by other methods known in the art, or administered to ex vivo cells. In some embodiments where the Wnt surrogate molecule is administered to ex vivo cells, these cells may be transplanted into a subject before, after or during administration of the Wnt surrogate molecule.

Wnt signaling is a key component of stem cell culture. For example, the stem cell culture media as described in WO2010/090513, WO2012/014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5). The Wnt surrogate molecules disclosed herein are suitable alternatives to Rspondin for use in these stem cell culture media, or may be combined with Rspondin.

Accordingly, in one embodiment, the disclosure provides a method for enhancing the proliferation of stem cells comprising contacting stem cells with one or more Wnt surrogate molecules disclosed herein. In one embodiment, the disclosure provides a cell culture medium comprising one or more Wnt surrogate molecules disclosed herein. In some embodiments, the cell culture medium may be any cell culture medium already known in the art that normally comprises Wnt or Rspondin, but wherein the Wnt or Rspondin is replaced (wholly or partially) or supplemented by Wnt surrogate molecule(s) disclosed herein. For example, the culture medium may be as described in as described in WO2010/090513, WO2012/014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5), which are hereby incorporated by reference in their entirety.

Stem cell culture media often comprise additional growth factors. This method may thus additionally comprise supplying the stem cells with a growth factor. Growth factors commonly used in cell culture medium include epidermal growth factor (EGF, (Peprotech), Transforming Growth Factor-alpha (TGF-alpha, Peprotech), basic Fibroblast Growth Factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), Hepatocyte Growth Factor (HGF) and Keratinocyte Growth Factor (KGF, Peprotech, also known as FGF7). EGF is a potent mitogenic factor for a variety of cultured ectodermal and mesodermal cells and has a profound effect on the differentiation of specific cells in vivo and in vitro and of some fibroblasts in cell culture. The EGF precursor exists as a membrane-bound molecule which is proteolytically cleaved to generate the 53-amino acid peptide hormone that stimulates cells. EGF or other mitogenic growth factors may thus be supplied to the stem cells. During culturing of stem cells, the mitogenic growth factor may be added to the culture medium every second day, while the culture medium is refreshed preferably every fourth day. In general, a mitogenic factor is selected from the groups consisting of: i) EGF, TGF-alpha, and KGF, ii) EGF, TGF-alpha, and FGF7; iii) EGF, TGF-alpha, and FGF; iv) EGF and KGF; v) EGF and FGF7; vi) EGF and a FGF; vii) TGF-alpha and KGF; viii) TGF-alpha, and FGF7; ix) or from TGF-alpha and a FGF. In certain embodiments, the disclosure includes a stem cell culture media comprising a Wnt surrogate molecule disclosed herein, e.g., optionally in combination with one or more of the growth factors or combinations thereof described herein.

These methods of enhancing proliferation of stem cells can be used to grow new organoids and tissues from stem cells, as for example described in WO2010/090513 WO02012/014076, Sato et al., 2011 (GASTROENTEROLOGY 2011; 141: 1762-1772) and Sato et al., 2009 (Nature 459, 262-5).

In some embodiments, the Wnt surrogate molecules are used to enhance stem cell regeneration. Illustrative stem cells of interest include but are not limited to: muscle satellite cells; hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neural stem cells (see Morrison et al. (1999) Cell 96: 737-749); embryonic stem cells: mesenchymal stem cells; mesodermal stem cells; liver stem cells; adipose-tissue derived stem cells, etc.

Other embodiments of the present invention relate, in part, to diagnostic applications for detecting the presence of cells or tissues expressing one or more Fzd receptors or LRP5 or LRP6 receptors. Thus, the present disclosure provides methods of detecting one or more Fzd receptor or LRP5 or LRP6 receptor in a sample, such as detection of cells or tissues expressing Fzd1. Such methods can be applied in a variety of known detection formats, including, but not limited to immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), whole-mount in situ hybridization (WISH), fluorescent DNA in situ hybridization (FISH), flow cytometry, enzyme immuno-assay (EIA), and enzyme linked immuno-assay (ELISA), e.g., by detecting binding of a Wnt surrogate molecule.

ISH is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., primary binding agent) to localize a specific DNA or RNA sequence in a portion or section of a cell or tissue (in situ), or if the tissue is small enough, the entire tissue (whole mount ISH). One having ordinary skill in the art would appreciate that this is distinct from immunohistochemistry, which localizes proteins in tissue sections using an antibody as a primary binding agent. DNA ISH can be used on genomic DNA to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts.

In various embodiments, the Wnt surrogate molecules described herein are conjugated to a detectable label that may be detected directly or indirectly. In this regard, an antibody “conjugate” refers to a Wnt surrogate molecule that is covalently linked to a detectable label. In the present invention, DNA probes, RNA probes, monoclonal antibodies, antigen-binding fragments thereof, and antibody derivatives thereof, such as a single-chain-variable-fragment antibody or an epitope tagged antibody, may all be covalently linked to a detectable label. In “direct detection”, only one detectable antibody is used, i.e., a primary detectable antibody. Thus, direct detection means that the antibody that is conjugated to a detectable label may be detected, per se, without the need for the addition of a second antibody (secondary antibody).

A “detectable label” is a molecule or material that can produce a detectable (such as visually, electronically or otherwise) signal that indicates the presence and/or concentration of the label in a sample. When conjugated to an antibody, the detectable label can be used to locate and/or quantify the target to which the specific antibody is directed. Thereby, the presence and/or concentration of the target in a sample can be detected by detecting the signal produced by the detectable label. A detectable label can be detected directly or indirectly, and several different detectable labels conjugated to different specific-antibodies can be used in combination to detect one or more targets.

Examples of detectable labels, which may be detected directly, include fluorescent dyes and radioactive substances and metal particles. In contrast, indirect detection requires the application of one or more additional antibodies, i.e., secondary antibodies, after application of the primary antibody. Thus, the detection is performed by the detection of the binding of the secondary antibody or binding agent to the primary detectable antibody. Examples of primary detectable binding agents or antibodies requiring addition of a secondary binding agent or antibody include enzymatic detectable binding agents and hapten detectable binding agents or antibodies.

In some embodiments, the detectable label is conjugated to a nucleic acid polymer which comprises the first binding agent (e.g., in an ISH, WISH, or FISH process). In other embodiments, the detectable label is conjugated to an antibody which comprises the first binding agent (e.g., in an IHC process).

Examples of detectable labels which may be conjugated to Wnt surrogate molecules used in the methods of the present disclosure include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, metal particles, haptens, and dyes.

Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescent protein (GFP) and analogues thereof, and conjugates of R-phycoerythrin or allophycoerythrin, inorganic fluorescent labels such as particles based on semiconductor material like coated CdSe nanocrystallites.

Examples of polymer particle labels include micro particles or latex particles of polystyrene, PMMA or silica, which can be embedded with fluorescent dyes, or polymer micelles or capsules which contain dyes, enzymes or substrates.

Examples of metal particle labels include gold particles and coated gold particles, which can be converted by silver stains. Examples of haptens include DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin. Examples of enzymatic labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase, β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase and glucose oxidase (GO). Examples of commonly used substrates for horseradishperoxidase include 3,3′-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), Benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphtol (CN), .alpha.-naphtol pyronin (.alpha.-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolylphosphate (BCIP), Nitro blue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), tetranitro blue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxyl-beta-D-galactoside/ferro-ferricyanide (BCIG/FF).

Examples of commonly used substrates for Alkaline Phosphatase include Naphthol-AS-B 1-phosphate/fast red TR (NABP/FR), Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR), Naphthol-AS-B1-phosphate/-fast red TR (NABP/FR), Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR), Naphthol-AS-B1-phosphate/new fuschin (NABP/NF), bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT), 5-Bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

Examples of luminescent labels include luminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur and phosphorous.

Detectable labels may be linked to the antibodies described herein or to any other molecule that specifically binds to a biological marker of interest, e.g., an antibody, a nucleic acid probe, or a polymer. Furthermore, one of ordinary skill in the art would appreciate that detectable labels can also be conjugated to second, and/or third, and/or fourth, and/or fifth binding agents or antibodies, etc. Moreover, the skilled artisan would appreciate that each additional binding agent or antibody used to characterize a biological marker of interest may serve as a signal amplification step. The biological marker may be detected visually using, e.g., light microscopy, fluorescent microscopy, electron microscopy where the detectable substance is for example a dye, a colloidal gold particle, a luminescent reagent. Visually detectable substances bound to a biological marker may also be detected using a spectrophotometer. Where the detectable substance is a radioactive isotope detection can be visually by autoradiography, or non-visually using a scintillation counter. See, e.g., Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (Humana Press, Totowa, N.J.).

The invention further provides kits for detecting one or more Fzd or LRP5/6 receptor or cells or tissues expressing one or more Fzd or LRP5/6 receptors in a sample, wherein the kits contain at least one antibody, polypeptide, polynucleotide, vector or host cell as described herein. In certain embodiments, a kit may comprise buffers, enzymes, labels, substrates, beads or other surfaces to which the antibodies of the invention are attached, and the like, and instructions for use.

All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

EXAMPLES Example 1 Illustrative Wnt Surrogate Molecule Formats

Wnt surrogates having a variety of different formats disclosed herein were made. These included the following illustrative formats, each comprising a binding domain (“binder”) that binds to one or more Fzd receptor and a binding domain (“binder”) that binds to an LRP5 and/or LRP6 receptor.

As shown in FIG. 1A, if a binder to one receptor is a Fab and to the other receptor is a Nab or scFv, they can be put together in several different configurations. In certain instances, the Fab binder first can be reformatted to a full IgG format, then the Nab binder can be fused to any of the 4 available termini of the IgG. For example, the Nab can be fused to the N-terminus of the IgG light chain (LC, the fusion will be referred to as NL, shown in top left), N-terminus of the IgG heavy chain (HC, the fusion will be referred to as NH, shown in top right), the C-terminus of LC (the fusion will be referred to as CL, shown in middle right), and the C-terminus of HC (the fusion will be referred to as CH, shown in middle left). The linkers and the length of the linkers between the IgG and the Nab can be varied. These four formats are bispecific and bivalent, they are bivalent binders toward each of the receptors. An alternative way to put the two binders together is the Hetero-Ig format where the Fab binder is presented as a half antibody, and the Nab is fused to the N-terminus of an Fc (shown in lower middle). The two halves may be brought together by mutations in the CH3 domain (such as the knobs-into-holes) that favor the formation of the heterodimer. The linker and its length between the Nab binder and the Fc can be varied. This format would be bispecific but monovalent toward each receptor. The Nab part of any of the formats described in this example can also be replaced by scFv fragments of binders as well.

As shown in FIG. 1B, if a binder to one receptor is a Fab and to the other receptor is also a Fab, they can be put together in several different configurations. In one approach, one Fab binder is first reformatted to a full IgG format (shown in top). The second Fab binder can be fused to the N-terminus of the IgG. The two HC can be fused together with a linker in between. The LCs can be fused or unfused. The linker and its length can be varied. This format is a bispecific and bivalent format. Alternatively, the second Fab binder LC can be fused to the HC of the IgG with a linker of various length in between. The second Fab binder HC can be fused or unfused to the LC of the IgG. A variation of this format has been called Fabs-in-tandem IgG (or FIT-Ig). In another approach, the two binders can be brought together as a Hetero-Ig by mutations in the CH3 domain that favors the heterodimer assembly, the two arms will each bind to one receptor (shown in bottom). This format is a bispecific and monovalent binder.

As shown in FIG. 1C, if a binder to one receptor is a Nab and to the other receptor is also a Nab, they can be put together in several different configurations. In the bispecific bivalent formats, in certain instances, the two Nab binders can be fused together in tandem (shown in top row) or fused to the two-different ends of the Fc (shown in middle row). The linker and its length between Nab and Nab or Nab and Fc can be varied. Alternatively, the two Nabs can be assembled together as Hetero-Ig to generate a bispecific and monovalent format (shown in bottom row). Similar to FIG. 1A, the Nab domains here can also be replaced by an scFv domain of a binder. In all the examples, the Nab and scFv can be mixed in certain combinations as well.

As shown in FIG. 1D, the binders against Fzd and LRP can also be linked together in a diabody (or DART) configuration. The diabody can also be in a single chain configuration. If the diabody is fused to an Fc, this will create a bivalent bispecific format. Without fusion to Fc, this would be a monovalent bispecific format.

A number of Wnt surrogates representing different configurations were produced. These included the Wnt surrogates described in Table 3. These illustrative Wnt surrogates include one, two or three polypeptides, the sequences of which are provided as Sequence 1, Sequence 2, and/or Sequence 3. The sequence may include a leader peptide sequence, a Nab sequence, a linker, and/or a heavy or light chain sequence. Annotated sequences are provided in FIG. 19 , in which the leader peptide sequence is italicized, the linker sequence is underlined, the Nab sequence is shown in bold, and the remaining sequence is the heavy chain or light chain sequence. The Fzd binder IDs and LRP binder IDs correspond to the clone numbers provided in Tables 1A-B and 2A-B for various Fzd binding or LRP5/6 binding antibodies or antigen-binding fragments thereof.

The Wnt surrogates beginning with “R2M3” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 001S-A04. The first six Wnt surrogates beginning with “18R5” in Table 3 include different LRP6 binding domains fused to the N-terminus of the anti-Fzd antibody or antigen-binding fragment thereof named 18R5. The Wnt surrogates beginning with “1R” include the anti-LRP6 antibody of antigen-binding fragment thereof named “009S-E04” fused to the N-terminus of different anti-Fzd antibodies or antigen-binding fragment thereof. For “R2M3-26CH,” the LRP6 binding region is fused to the C-terminus of the Fzd binding region. For “R2M3-26NH,” the LRP6 binding region is fused to the N-terminus of the Fzd binding region. For “R2M3-26CL,” the LRP6 binding region is fused to the C-terminus of the Fzd binding region. For “R2M3-26NL,” the LRP6 binding region is fused to the N-terminus of the Fzd binding region. For “R2M3-26Fab” and “R2M3-32Fab,” the LRP6 binding region is fused to the N-terminus of the Fzd binding region. For “Hetero-Ig,” the LRP6 binding region is fused to the N-terminus of human Fc_hole, and paired with Fzd binder light chain and heavy chain human IgG1_knob. The Wnt surrogates beginning with “17SB9” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 017S-B09. The Wnt surrogates beginning with “1R-C07” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 001S-B03. The Wnt surrogates beginning with “R2M13” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 004S-G06. The Wnt surrogates beginning with “3SD10” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 003S-D10. The Wnt surrogates beginning with “4SD1” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 004S-D01. The Wnt surrogates beginning with “14SB6” include different LRP6 binding domains fused to the N-terminus of a light chain region of the anti-Fzd antibody or antigen-binding fragment thereof named 014S-B06.

TABLE 3 Wnt Surrogate Sequences Fzd LRP Sequence 1 Sequence 2 Sequence 3 Name binder ID binder ID SEQ ID NO SEQ ID NO SEQ ID NO R2M3-23 001S-A04 009S-B04 89 95 N/A R2M3-26 001S-A04 009S-E04 90 95 N/A R2M3-28 001S-A04 009S-G04 91 95 N/A R2M3-29 001S-A04 009S-H04 92 95 N/A R2M3-31 001S-A04 013S-G04 93 95 N/A R2M3-32 001S-A04 013S-H04 94 95 N/A 18R5-5 18R5 008S-G01 96 104 N/A 18R5-7 18R5 008S-C02 97 104 N/A 18R5-8 18R5 008S-D02 98 104 N/A 18R5-9 18R5 008S-E02 99 104 N/A 18R5-26 18R5 009S-E04 100 104 N/A 18R5-28 18R5 009S-G04 101 104 N/A 18R5-31 18R5 013S-G04 102 104 N/A 18R5-32 18R5 013S-H04 103 104 N/A 1R-B05-26 001S-E02 009S-E04 105 111 N/A 1R-C01-26 001S-B01 009S-E04 106 112 N/A 1R-C07-26 001S-B03 009S-E04 107 113 N/A 1R-E06-26 001S-H02 009S-E04 108 114 N/A 1R-G05-26 001S-G02 009S-E04 109 115 N/A 1R-G06-26 001S-A03 009S-E04 110 116 N/A R2M3-26CH 001S-A04 009S-E04 125 117 N/A R2M3-26NH 001S-A04 009S-E04 125 118 N/A R2M3-26CL 001S-A04 009S-E04 119 2254 N/A R2M3-26NL 001S-A04 009S-E04 120 2254 N/A R2M3-26Fab 001S-A04 009S-E04 120 122 N/A R2M3-26F(ab’)2 001S-A04 009S-E04 120 2252 R2M3-32Fab 001S-A04 013S-H04 123 122 N/A R2M3-26Hetero-Ig 001S-A04 009S-E04 125 126 127 26-17SB9 017S-B09 009S-E04 128 N/A N/A 26:Fc:17SB9-criss-cross 017S-B09 009S-E04 2192 2193 N/A 26:5:17SB9:Fc 017S-B09 009S-E04 2194 N/A N/A 26:10:17SB9:Fc 017S-B09 009S-E04 2195 N/A N/A 26:15:17SB9: Fc 017S-B09 009S-E04 2196 N/A N/A 17SB9:Fc:26 017S-B09 009S-E04 2197 N/A N/A 26:Fc:26+17SB9:Fc:17SB9 017S-B09 009S-E04 2198 2199 N/A 1R-C07-3 001S-B03 008S-D01 134 113 N/A 1R-C07-36 001S-B03 013S-D05 135 113 N/A R2M13-3 004S-G06 008S-D01 136 153 N/A R2M13-26 004S-G06 009S-E04 137 153 N/A R2M13-36 004S-G06 013S-D05 138 153 N/A R2M3-3 001S-A04 008S-D01 139 2254 N/A R2M3-36 001S-A04 013S-D05 140 2254 N/A 3SD10-3 003S-D10 008S-D01 141 154 N/A 3SD10-26 003S-D10 009S-E04 142 154 N/A 3SD10-36 003S-D10 013S-D05 143 154 N/A 4SD1-3 004S-D01 008S-D01 144 155 N/A 4SD1-26 004S-D01 009S-E04 145 155 N/A 4SD1-36 004S-D01 013S-D05 146 155 N/A 14SB6-3 014S-B06 008S-D01 147 156 N/A 14SB6-26 014S-B06 009S-E04 148 156 N/A 14SB6-36 014S-B06 013S-D05 149 156 N/A R2M9-3 003S-E07 008S-D01 150 157 N/A R2M9-26 003S-E07 009S-E04 151 157 N/A R2M9-36 003S-E07 013S-D05 152 157 N/A 18R5:5:1115.3:Fc 18R5 1115.3 2200 N/A N/A 18R5:10:1115.3:Fc 18R5 1115.3 2201 N/A N/A 18R5:15:1115.3:Fc 18R5 1115.3 2202 N/A N/A 1115.3:5:18R5:Fc 18R5 1115.3 2203 N/A N/A 1115.3:10:18R5:Fc 18R5 1115.3 2204 N/A N/A 1115.3:15:18R5:Fc 18R5 1115.3 2205 N/A N/A 18R5:5:YW211.31.57:Fc 18R5 YW211.31.57 2206 N/A N/A 18R5:10:YW211.31.57:Fc 18R5 YW211.31.57 2207 N/A N/A 18R5:15:YW211.31.57:Fc 18R5 YW211.31.57 2208 N/A N/A YW211.31.57:5:18R5:Fc 18R5 YW211.31.57 2209 N/A N/A YW211.31.57:10:18R5:Fc 18R5 YW211.31.57 2210 N/A N/A YW211.31.57:15:18R5:Fc 18R5 YW211.31.57 2211 N/A N/A 18R5:Fc:1115.3 18R5 1115.3 2212 N/A N/A 1115.3:Fc:18R5 18R5 1115.3 2213 N/A N/A 18R5:Fc:YW211.31.57 18R5 YW211.31.57 2250 N/A N/A YW211.31.57:Fc:18R5 18R5 YW211.31.57 2267 N/A N/A 421.1-R2M3 cp 001S-A04 421.1 2214 2215 2216 1RC07:5:10SA7 cp 1RC07 10SA7 2217 2218 2219 1RC07:10:10SA7 cp 1RC07 10SA7 2217 2218 2222 1RC07:15:10SA7 cp 1RC07 10SA7 2217 2218 2225 1RC07:5:10SG7 cp 1RC07 10SG7 2217 2227 2228 1RC07:10:10SG7 cp 1RC07 10SG7 2217 2227 2231 1RC07:15:10SG7 cp 1RC07 10SG7 2217 2227 2234 10SG7:5:1RC07 cp 1RC07 10SG7 2227 2217 2237 10SG7:10:1RC07 cp 1RC07 10SG7 2227 2217 2240 10SG7:15:1RC07 cp 1RC07 10SG7 2227 2217 2243 1RC07:5:10SA7 L->H 1RC07 10SA7 2244 2245 2246 1115.3:5:R2M3 L->H 001S-A04 1115.3 2247 125 2248 1115.3:10:R2M3 L->H 001S-A04 1115.3 2247 125 2249 10SG11-1RC07 1RC07 10SG11 2252 2253 N/A 18R5:5:1115.3:His 18R5 1115.3 2255 N/A N/A 18R5:10:1115.3:His 18R5 1115.3 2256 N/A N/A 18R5:15:1115.3:His 18R5 1115.3 2257 N/A N/A 1115.3:5:18R5:His 18R5 1115.3 2258 N/A N/A 1115.3:10:18R5:His 18R5 1115.3 2259 N/A N/A 1115.3:15:18R5:His 18R5 1115.3 2260 N/A N/A 18R5:5:YW211.31.57:His 18R5 YW211.31.57 2261 N/A N/A 18R5:10:YW211.31.57:His 18R5 YW211.31.57 2262 N/A N/A 18R5:15:YW211.31.57:His 18R5 YW211.31.57 2263 N/A N/A YW211.31.57:5:18R5:His 18R5 YW211.31.57 2264 N/A N/A YW211.31.57:10:18R5:His 18R5 YW211.31.57 2265 N/A N/A YW211.31.57:15:18R5:His 18R5 YW211.31.57 2266 N/A N/A

Example 2 Characterization of a Wnt Surrogate Molecule, R2M3-26

The R2M3-26 molecule consists of a Fzd binder (R2M3) and a LRP6 binder (26). The LRP6 binder 26 was fused to the N-terminus of R2M3 LC with a 5-amino acid linker as depicted in FIG. 2A. R2M3 was in the form of an IgG. The protein was purified by Protein A affinity column followed by a size-exclusion-chromatography (SEC) step. The absorbance trace from the SEC and the SDS-PAGE gels of the SEC fractions were shown in FIG. 2B. The ability of R2M3-26 to activate canonical Wnt signaling was tested in a Wnt responding 293 reporter cell line (293STF). The 293STF reporter activity traces across the SEC fractions were shown in FIG. 2B, the peak of the reporter activity correlated with the peak of the proteins. The peak fraction was further characterized by a dose response in 293STF cells in the absence and presence of R-spondin (FIG. 2D). R2M3-26 induced reporter activity in a dose dependent manner and was enhanced by the presence of R-spondin similar to a natural Wnt ligand, while R2M3 IgG alone without the attachment of the LRP binding arm did not induce reporter activity. The ability of R2M3-26 to interact with its target, a Fzd1 ECD, was performed in Octet interaction assay (FIG. 2C), and the results showed that the fusion of the LRP6 binding arm, 26, did not affect R2M3 interaction with its target, Fzd.

Example 3 Characterization of a Wnt Surrogate Molecule, R2M3-32

The R2M3-32 molecule consists of a Fzd binder (R2M3) and a LRP6 binder (32). The LRP6 binder 32 was fused to the N-terminus of R2M3 LC with a 5-amino acid linker as depicted in FIG. 3A. R2M3 was in the form of an IgG. The protein was purified by Protein A affinity column followed by a size-exclusion-chromatography (SEC) step. The absorbance trace from the SEC and the SDS-PAGE gels of the SEC fractions were shown in FIG. 3B. The ability of R2M3-32 to activate canonical Wnt signaling was tested in a Wnt responding 293 reporter cell line (293STF). The 293STF reporter activity traces across the SEC fractions were shown in FIG. 3B, the peak of the reporter activity correlated with the peak of the proteins. The peak fraction was further characterized by a dose response in 293STF cells in the absence and presence of R-spondin (FIG. 3D). R2M3-32 induced reporter activity in a dose dependent manner and was enhanced by the presence of R-spondin, while R2M3 IgG alone without the attachment of the LRP binding arm did not induce reporter activity. The ability of R2M3-32 to interact with its target, a Fzd1 extracellular domain (ECD), was performed in Octet interaction assay (FIG. 3C). The results showed that the fusion of the LRP6 binding arm, 32, did not affect R2M3 interaction with its target, Fzd.

Example 4 R2M3-26 and R2M3-32 Activities can be Inhibited by Soluble Fzd ECD and by R2M3 IgG Alone without the LRP Binding Arm

The ability of soluble Fzd extracellular domain (ECD) or R2M3 IgG alone to inhibit Wnt surrogates was determined using the 293STF reporter assay. The Fzd1 ECD-Fc or R2M3 IgG was titrated into the 293STF reporter assay, at a fixed concentration of R2M3-26 or R2M3-32. In a dose dependent manner, both Fzd1 ECD-Fc and R2M3 IgG inhibited R2M3-26 (FIG. 4A) and R2M3-32 (FIG. 4B) induced reporter signaling, while the negative control molecule, Fc alone had no impact.

Example 5 Characterization of R2M3-LRP6 Binder Fusions in 293, Huh7, A375, BNL.CL2 Wnt Dependent Reporter Assays

The Fzd binder, R2M3, was fused to additional LRP6 binders, 23, 25, 26, 27, 28, 29, 31, 32, 33, and 36. The LRP6 binders were Nab and were fused to the N-terminus of R2M3 LC with a 5-amino acid linker. These proteins were purified by Protein A affinity column followed by a SEC step. The fusion proteins were tested in Wnt dependent reporter assays in 293, Huh7, A375, and BNL.CL2 cell lines, and activated Wnt signaling to various levels. R2M3 was also fused to two non-LRP6 binder Nabs, 24 and 34, in the same format as the Lrp6 binders. These two non-binders displayed no activity in Wnt dependent 293 reporter assay (FIG. 5 ), suggesting that Wnt activities observed with R2M3 fusions to 23, 25, 26, 27, 28, 29, 31, 32, 33, and 36 are dependent on the presence of both Fzd and Lrp mimicking the nature ligand function.

Example 6 Characterization of 18R5-LRP6 Binder Fusions in 293, A375, and BNL.CL2 Wnt Dependent Reporter Assays

The Fzd binder, 18R5, was fused to LRP6 binders, 26, 28, 31, 32. The LRP6 binders were Nab and were fused to the N-terminus of 18R5 LC with a 5-amino acid linker. These proteins were purified by Protein A affinity column followed by a SEC step. The fusion proteins were tested in Wnt dependent reporter assays in 293, A375, and BNL.CL2 cell lines and demonstrated ability to activate Wnt signaling (FIG. 6 ).

Example 7 Characterization of 18R5-LRP5 Binder Fusions in 293 Wnt Dependent Reporter Assays

The Fzd binder, 18R5, was fused to LRP5 binders, 5, 7, 8, 9. The LRP5 binders were Nab and were fused to the N-terminus of 18R5 LC with a 5-amino acid linker. These proteins were purified by Protein A affinity column followed by a SEC step. The fusion proteins were tested in a Wnt dependent reporter assays in 293 cells and were able to activate Wnt signaling (FIG. 7 ).

Example 8 Characterization of Various Fzd Binders-LRP6 Binder 26 Fusions in 293 Wnt Dependent Reporter Assays

The various Fzd binders, 1R-B05, 1R-C01, 1R-C07, 1R-E01, 1R-E06, 1R-G05, 1R-G06, 1R-H04, in IgG format were fused to LRP6 binders, 26. The LRP6 binder Nab was fused to the N-terminus of various Fzd binder LC with a 5-amino acid linker. These proteins were purified by Protein A affinity column followed by a SEC step. The SDS-PAGE gel analysis of the SEC peak fractions were shown in FIG. 8A. The fusion proteins were tested in a Wnt dependent reporter assays in 293 cells in the presence of Rspo and were able to activate Wnt signaling (FIG. 8B).

Example 9 SAR Analysis of the IgG-Nab Fusion Format

SAR analysis of the IgG-Nab fusions was performed by rotating the attachment location of the Nab to the different termini of the IgG HC or LC as depicted in FIG. 1A. CH indicates attaching the Nab to the C-terminus of heavy chain; NH indicates attaching the Nab to the N-terminus of heavy chain; CL indicates attaching the Nab to the C-terminus of light chain; NL indicates attaching the Nab to the N-terminus of light chain. Three pairs of IgG-Nab fusions SARs were shown, the pairs were between R2M3 and 26, between R2M3 and 32, and between 18R5 and 26. The assays were performed on Wnt responsive 293 reporter cells in the presence of Rspo and activated Wnt signaling to various levels (FIG. 9 ). These results demonstrate that the attachment location of the fusion and the geometry between the Fzd and LRP binding domains play roles in the ability of the Wnt surrogates to activate Wnt signaling.

Example 10 Characterization of R2M3-26 in the Fab Format

The molecule R2M3-26 Fab consists of a Fzd binder (R2M3) and a LRP6 binder (26). The LRP6 binder 26 was fused to the N-terminus of R2M3 LC with a 5-amino acid linker as depicted in FIG. 10A. R2M3 was in the form of a Fab. The protein was purified by Ni-NTA affinity column followed by a size-exclusion-chromatography (SEC) step. The absorbance trace from the SEC and the SDS-PAGE gels of the SEC fractions are shown in FIG. 10B. The ability of R2M3-26 as a Fab format to activate canonical Wnt signaling was tested in a Wnt responding 293 reporter cells (293STF). The 293STF reporter activity traces across the SEC fractions are shown in FIG. 10B. Unlike when R2M3 was in the IgG format shown in FIG. 2 , the peak of the reporter activity from the R2M3 in the Fab format did not correlate with the peak of the proteins. These results suggest that R2M3-26 fusion in the Fab format is ineffective in inducing canonical Wnt signaling as detected by a reporter assay.

Example 11 Characterization of R2M3-32 in the Fab Format

The molecule R2M3-32 Fab consists of a Fzd binder (R2M3) and a LRP6 binder (32). The Lrp6 binder 32 was fused to the N-terminus of R2M3 LC with a 5-amino acid linker as depicted in FIG. 11A. R2M3 was in the form of a Fab. The protein was purified by Ni-NTA affinity column followed by a size-exclusion-chromatography (SEC) step. The absorbance trace from the SEC and the SDS-PAGE gels of the SEC fractions were shown in FIG. 11B. The ability of R2M3-32 as a Fab format to activate canonical Wnt signaling was tested in a Wnt responding 293 reporter cells (293STF). The 293STF reporter activity traces across the SEC fractions are shown in FIG. 11B. Unlike when R2M3 was in the IgG format shown in FIG. 3 , the peak of the reporter activity from the R2M3 in the Fab format did not correlate with the peak of the proteins. These results suggest that R2M3-32 fusion in the Fab format is ineffective in inducing canonical Wnt signaling as detected by a reporter assay.

Example 12 Characterization of R2M3-26 in the Heterolg Format

The molecule R2M3-26 Heterolg consists of a Fzd binder (R2M3) and a LRP6 binder (26) as depicted in FIG. 12A and described in FIG. 1A. The protein was purified by Protein A affinity column followed by a size-exclusion-chromatography (SEC) step. The peak fraction from the SEC column was tested in a dose response in the Wnt responsive 293STF reporter cells in the absence or presence of R-spondin (FIG. 12B). Compared to R2M3-26 in the IgG format (as described in FIG. 2 ), R2M3-26 Heterolg was ineffective in inducing canonical Wnt signaling as detected in the 293 reporter assay.

Example 13 Characterization of 26-17SB9 in the Nab-Nab Format

The molecule 26-17SB9 Nab-Fc-Nab consists of a LRP6 binder (26) and a Fzd binder (17SB9) as depicted in FIG. 13A and described in FIG. 1C. The protein was purified by Protein A affinity column followed by a size-exclusion-chromatography (SEC) step. The peak fraction from the SEC column was tested in a dose response in the Wnt responsive 293STF reporter cells in the absence or presence of R-spondin (FIG. 13B). 26-17SB9 in the Nab-Fc-Nab format induced canonical Wnt signaling as detected in the 293 reporter assay.

Additional combinations of 26 and 17SB9 were also constructed (FIG. 13C) and tested in 293 reporter assays. As shown in FIGS. 13D and 13E, these various combinations where 26 and 17SB9 were arranged in different tandem formats or on different ends of the Fc fragment all activated Wnt signaling to various levels in the presence of 20 nM R-spondin

Example 14 Characterization of 18R5-LRP6 Binder Fusions in Tandem scFv Formats

The Fzd binder 18R5, the LRP6E1E2 binder 1115.3 (as described in PCT Publication WO2009/064944), and the LRP6E3E4 binder YW211.31.57 (as described in PCT Publication WO2011/119661) were converted into scFv format. 1115.3_scFv or YW211.31.57_scFv is assembled to the N-terminus of 18R5_scFv with a 5, 10 or 15-amino acid linker and 18R5_scFv C-terminus is fused to a human Fc domain. In another set of examples, 1115.3_scFv or YW211.31.57_scFv is assembled to the C-terminus of 18R5 scFv with a 5, 10 or 15-amino acid linker and human Fc domain is fused to the C-terminus of LRP binders. These formats are depicted in FIG. 14G left panel. In another example, 18R5_scFv and LRP binder, 1115.3_scFv, or LRP binder, YW211.31.57_scFv were fused to the two ends of a human Fc domain (as depicted in FIG. 14G right panel). These proteins were purified by Protein A affinity column followed by a SEC step.

The fusion proteins were tested in a Wnt dependent reporter assays in 293 cells. 18R5_scFv-1115.3_scFv-Fc and 1115.3_scFv-18R5_scFv-Fc with 5, 10 or 15-mer linker were able to activate Wnt signaling (FIGS. 14A and 14B). 18R5_scFv-YW211.31.57_scFv-Fc and YW211.31.57_scFv-18R5_scFv-Fc with different linkers activated Wnt signaling (FIGS. 14C and 14D). In addition, 18R5_scFv and 1115.3_scFv or YW211.31.57 fused to the two ends of Fc also activated Wnt signaling (FIGS. 14E and F). While all of these scFv formats activated Wnt signaling, potency and overall maximal efficacy may differ depends on the binder combination, linker length, and relative orientation.

In another example, 1115.3_scFv or YW211.31.57_scFv is assembled to the N-terminus or C-terminus of 18R5_scFv with a 5, 10 or 15-amino acid linker without the further fusion of Fc, to create a bispecific but monovalent binding to each of Fzd or LRP. As shown in FIG. 14H, the 1115.3_scFv and 18R5_scFv fusions were in effective in activating Wnt signaling in the presence of 20 nM R-spondin in 293 reporter cells.

Example 15 Generation of Wnt Surrogate Molecules in the Fab-IgG Format

Wnt mimetic or surrogate molecules can be generated in various formats when both the FZD and LRP binders are Fabs. Various approaches, such as charge paring, “knobs-in-holes”, crossover of the heavy and light chains of Fabs, can be employed to ensure proper heavy and light chain pairing. Two examples are given below.

-   -   1. Charge-pairing (cp) approach for Fab-on-IgG format: The heavy         chain (VH-CH1) domain of an anti-LRP6 Fab, through a linker of         5, 10, or 15-mer amino acids, was fused in tandem with the         N-terminus of the heavy chain (VH-CH1-CH2-CH3) of an anti-FZD         binder. Both VH-CH1 domains of anti-LRP6 and anti-FZD contain         three amino acid mutations (Q39D, Q105D, S183K in the anti-LRP6         Fab; Q39K, Q105K, S183E in anti-FZD Fab) each for proper paring         with their own partner light chains, which also contain three         complementary amino acid mutations (Q38K, A/S43K, S176E in         anti-LRP6 light chain; Q38D, A/S43D, S176K in the anti-FZD light         chain). The order of the anti-LRP6 and anti-FZD Fabs could be         reversed, where the anti-FZD binder is a Fab and is fused to         anti-LRP binder which is in IgG format (FIG. 15A).     -   2. HC-LC cross over approach for Fab-on-IgG format: The light         chain (VL-CL) domains of anti-LRP6 binder was, through a linker         of 5, 10, or 15-mer amino acids, fused in tandem with the         N-terminus of the heavy chain (VH-CH1-CH2-CH3) of an anti-FZD         binder. The second construct was VH-CH1 of the anti-LRP6 binder         and the third construct was VL-CL of the anti-FZD binder.         Similar to the example above, the order of the anti-LRP6 and the         anti-FZD binders could be reversed, where anti-FZD binder Fab is         fused to the N-terminus of the anti-LRP binder which is in IgG         format (FIG. 15A).

Several different pairs of LRP and FZD binders were assembled in these formats and tested in a Wnt responding 293 reporter cell line (293STF). As an example, the anti LRP6E1E2 binder 421.1 (as described in PCT Publication WO2009/064944) was fused to the N-terminus of anti-FZD binder, R2M3, using the charge paring approach to generate, 421.1-R2M3 cp. 421.1-R2M3 cp dose-dependently activated Wnt signaling in the 293 reporter assay (FIG. 15B). An anti-FZD binder, 1RC07, was fused to the N-terminus of an anti-LRP binder, 10SA7, with 5, 10, or 15-mer linkers. All three fusion proteins activated Wnt signaling (FIG. 15C). The anti-FZD binder, 1RC07, was further fused with the anti-LRP binder, 10SG7, either with 1RC07 in the Fab format fused to the N-terminus of 10SG7 in IgG format, or in the reverse order where 10SG7 as Fab fused to the N-terminus of 1RC07 as IgG with either 5, 10, or 15-mer amino acid linkers. All fusion molecules activated Wnt signaling while some preference of orientation and linker length was observed (FIGS. 15D and 15E).

The HC-LC crossover Fab-IgG format was also tested. The anti-FZD binder 1RC07 LC was fused to the N-terminus of the anti-LRP6 binder 10SA7 HC to generate 1RC07-5: 10SA7 L→H. The LC of anti-LRP6E1E2 binder 1115.3 (as described in PCT Publication WO2009/064944) was fused to the N-terminus of the anti-FZD binder R2M3 HC with 5 or 10-mer linkers to generate 1115.3:5:R2M3 L→H or 1115.3:10:R2M3 L→H, respectively. These molecules also activated Wnt signaling (FIGS. 15F and 15G).

Example 16 Characterization of R2M3-26 in the F(ab′)2 Format

R2M3-26 IgG1 was digested by IDES (Promega, WI) at 37° C. for 2 hours. Vast majority of the digested product was R2M3-26F(ab′)2 (FIG. 16A), some partially digested product with one Fab still attached to Fc (designated as R2M3-26F(ab′)2-Fc here) was also detected, and no uncleaved R2M3-26 was detected. The cleaved product was purified by anti-Lambda resin to remove the Fc fragment, then a SEC polishing was followed to separate the R2M3-26F(ab′)2 from R2M3-26F(ab′)2-Fc. The SDS-PAGE gel of the final purified protein is shown in FIG. 16B. The R2M3-26F(ab′)2 activity was measured in STF assay in HEK293 cells. R2M3-26F(ab′)2 was able to activate Wnt signaling (FIG. 16C).

Example 17 Characterization of Additional Wnt Surrogate Molecules

The FZD binders were fused to LRP binders. The LRP5 or 6 binders were Nabs (or VHHs) in this example and were fused to the N-terminus of FZD binders LC with a 5-amino acid linker (as represented in FIG. 17A). These proteins were purified by Protein A affinity column followed by a SEC step. The purified proteins were tested in Wnt dependent reporter assay in 293 cells (FIG. 17B, C, D, H), or 293 cells co-transfected with FZD4 expression construct (FIG. 17E, F), or 293 cells co-transfected with FZD9 expression construct (FIG. 17G) in the presence of 20 nM R-spondin. These molecules activated Wnt signaling with varying levels of potency and efficacy.

Example 18 Characterization of 10SG11-1RC07 in the 2Fv-Ig Format

The molecule 10SG11-1RC07 consists of an N-terminal LRP binder (10SG11) and a Fzd binder (1RC07). The Fv of 10SG11 was fused to the N-terminus of 1RC07 with a 5-amino acid linker as depicted in FIG. 18A. 1RC07 was in the form of an IgG1 with the Fc mutations L234A/L235A/P329G. The protein was purified by a Protein A affinity column followed by an SEC step. The fusion protein was tested in Wnt dependent reporter assays in 293 cell lines and demonstrated the ability to activate Wnt signaling (FIG. 18B-C)

Example 19 In Vivo PK/PD Characterization of R2M3-26

Six-week old C57Bl/6J male mice were obtained from Jackson Laboratories (Bar Harbor, Me., USA) and were housed 3 per cage. All animal experimentation was in accordance with the criteria of the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences. Protocols for animal experimentation were approved by the Surrozen Institutional Animal Care and Use Committee. Mice were acclimatized a minimum of two days prior to initiating experiments. Mice had unlimited access to purified, laboratory-grade acidified water and were fed ad libitum (2018 Teklad global 18% protein rodent diet) Mice were kept 12/12-hour light/dark cycle in a 30% to 70% humidity environment and room temperature ranging from 20° C. to 26° C.

For the pharmacokinetic (PK) study (FIG. 20A), n=3 per group was used. Mice were dosed with R2M3-26 (with effectorless Fc mutations) at 1 mg/kg (10 ml/kg in saline) either using intravenous (IV) or intraperitoneal (i.p.) injections. Mice were anesthetized with isoflurane and blood was removed from the retro-orbital plexus, tail vein or heart at 10 minutes, 30 minutes, 1, 4, 24, 72 or 144 hours after injection. Blood was allowed to coagulate at room temperature, followed by centrifugation for 7 minutes at 8,000 g. The serum was removed and stored at −20° C. until the measurement of serum R2M3-26 concentrations by ELISA with Anti Human IgG Fc Fragment (Jackson Immuno Research Labs NC9747692).

For the pharmacodynamic (PD) study (FIG. 20B), n=6 per group was used. Mice were injected i.p. with R2M3-26 at the indicated doses (10 ml/kg in saline). Control mice received saline only. Eight hours later, mice were anesthetized with isoflurane and the blood was collected by cardiac puncture. Blood was allowed to coagulate at room temperature, followed by centrifugation for 7 minutes at 10,000 g. The serum was removed and stored at −20° C. until the measurement of serum R2M3-26 concentrations by ELISA. A portion of the left liver lobe was snap-frozen in liquid nitrogen and stored at −80° C. for RNA analysis. RNA was extracted from liver samples using the MagMAX™ mirVana™ Total RNA Isolation Kit (ThermoFisher, A27828). cDNA was produced using the high-Capacity cDNA Reverse Transcription Kit (ThermoFisher, 43-688-14). Axin2 mRNA expression was measured by using TaqMan® Fast Advanced Master Mix (ThermoFisher, 4444963) and the Mm00443610_m1 Axin2 Probe (Thermofisher, 4331182).

These studies showed that R2M3-26 was stable, highly bioavailable and active in vivo, as shown by the induction of Axin2 mRNA expression.

Example 20 In Vivo Bone Model and Characterization of AAV-Delivered Wnt Surrogates

In vivo experiments were conducted by infecting mice with an AAV vector that expressed Flag- and His-tagged 18R5-DKK1c protein (AAV-18R5-DKK1c-FlagHis). 18R5-DKK1c is a fusion protein containing the frizzled binding antibody, 18R5, in scFv format, fused to DKK1c, as described in PCT Publication WO2016/040895, e.g., FIG. 5 . Control mice were treated with vehicle only, romosozumab, an AAV vector that expressed green fluorescent protein (GFP) (AAV-CAG-GFP), or an AAV vector that expressed a fusion protein comprising an anti-GFP scFv fused to a mutant DKK1c (AAV-ScFv (anti-GFP)-DKK1cF234K-Flag-His). 28 days after infection, animals were sacrificed and bone mineral density, bone volume and other characteristics were measured. As shown in FIGS. 21A-21E, systemic expression of 18R5-DKK1c resulted in significantly increased bone mineral density as early as 14 days of 18R5-DKK1c systemic expression, as determined by dual X-ray absorptiometry (DEXA) scan. Systemic expression of 18R5-DKK1c increased bone mineral density (BMD) as measured by DEXA scan (FIG. 21A) and increased levels of the serum P1NP bone formation marker in naïve mice (FIG. 21C). Serum levels of AAV-ScFv (anti-GFP)-DKK1cF234K and 18R5-DKK1c were detected in the serum and found to be well above the in vitro determined EC50 (FIG. 21B). AAV-CAG-GFP and AAV-ScFv (anti-GFP)-DKK1cF234K were negative controls. Romosozumab was a positive control, and vehicle only was a negative control. 18R5-DKK1c also increase bone density in lumbar vertebra and whole body, as shown in FIGS. 20D and 20E, wherein * indicates P value<0.05 and ** indicates P value<0.0001. 18R5-DKK1c expression through AAV also increased bone volume in tibia and femur and cortical thickness in femur mid-diaphysis at 28 days after treatment in naïve mice as measured by micro CT, as shown in FIGS. 22A-22D, wherein **** indicates P value<0.0001.

Systemic expression of 18R5-DKK1c resulted in significantly increased mineral apposition rate from baseline to single label in last 8 days, as shown in FIGS. 23A and 23B.

Systemic expression of 18R5-DKK1c also resulted in increased osteoblast numbers and decreased osteoclast numbers, as shown in FIGS. 24A-24D.

18R5-DKK1c treatment increased bone stiffness and ultimate load to fracture in biomechanical testing, suggesting improved resistance to fracture, as shown in FIGS. 25A-25C.

These studies demonstrated that systemic expression of 18R5-DKK1c using AAV increased bone mineral density (BMD) as measured by DEXA, and also showed that 18R5-DKK1c increased bone volume as measured by micro CT as early as 14 days of treatment. 18R5-DKK1c also increased cortical thickness 28 days after treatment. Systemic expression of 18R5-DKK1c resulted in significantly increased mineral apposition rate, increased osteoblast numbers, and decreased osteoclast numbers. It also increased bone stiffness and ultimate load to fracture, suggesting improved resistance to fracture.

Example 21 In Vivo Bone Model and Characterization of Wnt Surrogates Produced as Recombinant Proteins

In vivo experiments were conducted by treating mice with recombinantly produced R2M3-26 protein at various dosages via i.p. injection. Control mice were treated with vehicle only (negative control), romosozumab (positive control), anti-Beta-Galactosidase (negative control), or IgG2-anti-GFP (negative control). Bone mineral density (BMD) measured by DEXA and bone volume measured by micro CT were monitored longitudinally at indicated time points. Four weeks after treatment, animals were sacrificed and bone characteristics were measured. For single injection of R2M3-26, the experimental data was monitored and is shown for two weeks after treatment.

Treatment with recombinant R2M3-26 induced rapid and sustained increase of bone mineral density (BMD) and bone volume in naive mice, as shown in FIGS. 26A-26D. Both bone volume and BMD increased rapidly, suggesting resistance to fracture.

The ovariectomy induced osteoporosis model is a well-established high-hurdle model for determining the ability of an anabolic therapy to overcome the bone loss associated with hormone ablation (Zhou, S. et. al., Journal of Cellular Biochemistry, PMID: 11455579). Treatment with recombinant R2M3-26 treatment reversed bone loss in an ovariectomy-induced osteoporosis mouse model, as shown in FIGS. 27A-27C. Increased cortical thickness was observed in the trabecular regions, suggesting increased compressive strength. R2M3-26 treatment increased femur mid-diaphysis cortical bone thickness after 42 days as measured by micro CT, as shown by FIG. 27D. BMD was also increased by R2M3-26 as measured by DEXA, shown in FIG. 27E.

A single injection of R2M3-26 was sufficient to induce rapid bone formation and bone volume within one week, as shown in FIGS. 28A-28C, wherein * indicates P value<0.05.

High dose treatment with R2M3-26 and 1R-C07-26 rapidly and significantly increased bone volume and bone mineral density, and improved biomechanical strength of the bone (ultimate load to failure and stiffness), as shown in FIGS. 29A-29D. 1R-C07-26 showed a robust and significant effect on bone accrual that persisted through 28 days. Both R2M3-26 and 1R-C07-26 significantly increased the resistance to fracture after 28 days of treatment by biomechanical testing.

High dose treatment with R2M3-26 and 1R-C07-3 rapidly and significantly increased bone volume, bone mineral density, and cortical thickness after only 14 days of treatment, as shown in FIG. 30A-E. 1R-C07-3 at 10 mpk appeared more effective at increasing bone mass than any other treatment tested in this preclinical model.

These studies demonstrate that recombinant protein treatment can induce rapid and sustained increase of bone mineral density and bone volume in naive mice and mouse osteoporosis model. Both bone volume and bone mineral density (BMD) increased rapidly, suggesting resistance to fracture. IgG2-anti-GFP is a negative control. Anti-Beta Galactosidase (anti-βgal) was a negative control.

An additional experiment was done to determine systemic skeletal effects of Wnt surrogate molecules in an ovariectomy-induced model of osteoporosis. 057BL/6 females, 4 weeks old at time of ovariectomy, (n=8/group) were compared to sham surgery operated as well as age-matched naïve mice. Animals were injected i.p. with recombinant Wnt surrogate molecules 7 months after surgery and when the onset of osteoporosis was confirmed. Experimental groups included R2M3-26, 1RC07-3, anti-Bgal (Ab control), and vehicle (PBS). Sub-cutaneous injection of romosozumab was done to another cohort of mice for comparison. Animal were treated twice weekly, and followed for 4 weeks.

As shown in FIG. 31 , whole body bone mineral density (BMD) was measured weekly using dual-energy X-Ray absorptiometry (DEXA) and treatment with Wnt surrogate molecules can not only reverse but even surpass the total BMD seen in naïve or non-surgical animals. After 4 weeks of treatment, animals were assessed for the vertebral resistance to compression fracture.

Treatment with Wnt surrogate molecules significantly increased the vertebral resistance to compression fracture, as shown by fracture analysis (FIG. 32 ). 1RC07-3 most robustly increases the Max Force required to compressively fracture the vertebra.

The Einhorn fracture model (Bonnarens F, Einhorn T A. J Orthop Res. 1984; 2(1):97-101.PMID: 6491805) with delayed treatment with Wnt surrogate molecules was used to determine ability of this therapy to induce fracture healing. Delayed treatment with either 1RCO7-3 or R2M3-26 was tested to determine if either molecule was capable of contributing to increased fracture healing after a mid-traverse femoral fracture. C57BL/6 females, 16 weeks old at time of fracture (n=8/group) were used. The presence of a cartilaginous callus 2 weeks after fracture was confirmed in all animals before the start of treatment. With delayed treatment to allow for callus formation, a pure osteogenic signal could be elicited, with rapid mineralization of the already existing callus.

Animals were injected i.p. with recombinant Wnt surrogate molecules with the following experimental groups: R2M3-26, 1RCO7-3, anti-Bgal (Ab control), and vehicle (PBS). Sub-cutaneous injection of romosozumab was done to another cohort of mice for comparison. Animal were treated twice weekly, and followed for 6 weeks. Radiography was used to visualize changes in mineralization of the callus throughout the experiment (FIGS. 33A and B). An increase in the mineralization and size of the resulting callus with Wnt surrogate treatment is apparent at both 1 and 6 weeks of treatment. One week was sufficient to induce rapid mineralization that is predictive of rapid bone fracture healing and resistance to fracture. 1RCO7-3 appeared to induce mineralization to a greater extent than R2M3-26. Radiographs taken after 6 weeks of treatment show the persistence of the highly mineralized callus in the 1RCO7-3 group, while some of the fracture callus in the R2M3-26 group has diminished (FIGS. 33B and D).

Whole body DEXA was measured throughout the experiment to examine bone mineral density in not only the fractured femur, but also the contralateral, non-fractured, femur where the expected increases in bone mineral density occurred after treatment (FIGS. 33C and D). This provides reduced risk of secondary fracture in the already fractured limb and the appendicular skeleton. BMD of contralateral femur at 42 days are shown in FIG. 33C.

Following 6-weeks of treatment after the confirmation of callus, we scanned the femurs with micro-computed tomography and determined multiple parameters which have been associated with an increased resistance to fracture after healing. Callus tissue volume, the bone volume within that callus, and especially bone mineral content are all significantly increased within the region of interest examined (FIG. 33D). Qualitatively, the reconstructions show the prevalence of thick osteoid and mineral within the treated fracture. These parameters suggest a robust resistance to fracture and indicate that delayed treatment with Wnt surrogate molecules, after the spontaneous cartilaginous callus formation, can initiate a rapid and significant increase in bone formation.

In another experiment, the dosing schedule was tested to determine whether Wnt surrogate molecule therapy can induce a significant bone anabolic effect, how long that effect persists, and after washout, to determine how the bone responds to additional treatment. Systemic skeletal effects were compared with variable dosing of 1RCO7-3 on anabolic effect, washout, and redosing after establishing baseline. C57BL/6 females, 12 weeks old (n=8/group) were injected i.p. with recombinant Wnt surrogate molecules with the following experimental groups: two groups with 1 RCO7-3, one with anti-βgal (Ab control), and one with vehicle (PBS) on day 0 of the experiment. Sub-cutaneous injection of romosozumab was done to another cohort of mice on day 0 for comparison (romosozumab is an anti-sclerostin antibody (Saag et al., N Engl J Med. 2017 Oct. 12; 377(15):1417-1427; PMID:28892457) that may reverse the bone loss associated with osteoporosis). Animals in 1 RC07-3 treated groups had significant and rapid induction of bone formation by 14 days (FIG. 34 ). One group received a second injection at day 14 to determine if the bone anabolic effect could be further enhanced. Interestingly, regardless of treatment, all treatment effects were reversed and normalized after 35 days. A 2-week period was allowed to return to baseline levels prior to a second round of treatment. On day 49, a second round of treatment was done to all experimental groups. The Wnt surrogate treated animals responded rapidly, however not to the same magnitude as with the initial treatment (FIG. 34 ). For all groups, 5 weeks after the last injection, new bone formation ceased. This indicates that a single injection is capable of significantly increasing bone formation, however the anabolic effect is rapidly lost. This suggests that an anti-resorptive agent may be required in combination with Wnt surrogate therapy to maintain the anabolic effect.

The mechanism of action of romosozumab relies upon the stimulation of bone formation by removing an inhibitor (sclerostin) of endogenous Wnt signaling. An experiment was done treating animals with Wnt surrogate molecules plus romosozumab to determine if Wnt surrogate molecule treatment was capable of synergizing with romosozumab in a combination study.

C57BL/6 males, 10 weeks old (n=8/group) were injected i.p. with recombinant Wnt surrogate molecules in combination with romosozumab in the following experimental groups: 1RC07-3 (0.1 mpk), 1RCO7-3 (1 mpk), 1RCO7-3 (10 mpk), 1RCO7-3 (0.1 mpk)+romosozumab (25 mpk), 1RCO7-3 (1 mpk)+romosozumab (25 mpk), 1RCO7-3 (10 mpk)+romosozumab (25 mpk), romosozumab (25 mpk) alone, anti-Bgal (Ab control), and vehicle (PBS). Animals were treated twice weekly and followed for 3 weeks.

Whole body BMD was measured weekly and results are presented in FIG. 35 . Conclusions from this study are that endogenous romosozumab can stimulate additional bone growth in the presence of high dose 1 RC07-3. These data further suggest that peak anabolic action has not yet been reached with 10 mpk 1RCO7-3 treatments. These data also show that romosozumab can stimulate bone formation even in the presence of 1RCO7-3. Overall, This study shows that Wnt surrogate molecule treatment can synergize with romosozmab to enhance the bone anabolic effect after only 21 days of twice weekly treatment.

Changes in gene expression in whole bone were measured in a time course after Wnt surrogate molecule therapy in mice to assess how this therapy regulates expression of genetic markers related to proliferation and osteogenesis. C57BL/6 females, 13 weeks old (n=5/group) were injected i.p. with 1RCO7-3 or anti-Bgal (Ab control) once. Sub-cutaneous injection of romosozumab was done to another cohort of mice for comparison. Cohorts of animals were sacrificed at 8, 24, 48 and 120 hours after treatment, and tibia bones and serum were isolated and flash frozen for RNA extraction. ELISA was used to measure levels of therapeutic molecules in serum over the course of the experiment as described above (FIG. 36 ).

For purification of RNA from bone, the excised tibia from freshly sacrificed animals were processed as follows: the ends of the tibia were clipped to expose marrow cavity and marrow cavity was flushed with ice cold saline through 30 gauge needle, Steps were taken to ensure all muscle tissue and cartilage has been removed; bone appeared completely white with no red marrow component residual. Tibias were placed in 1.5 mL Eppendorf tubes and flash frozen in liquid nitrogen. To lyse, a single tissue lyser bead was placed in the tube with bone and Trizol was added directly to frozen bone and bead. Tissue lyser at high speed was used to completely homogenize. Homogenates were then subject to chloroform extraction to separate the nucleic acid phase. Further isolation and purification was carried out using an RNeasy mini kit (Qiagen).

RNA Isolated from tibias was tested for relative transcript levels of Runt Related Transcription Factor 2 (RunX2), Collagen Type I Alpha 1 Chain (Col1A1), Dentin Matrix Acidic Phosphoprotein 1 (Dmp1), Alkaline phosphatase (Alp), Receptor activator of nuclear factor kappa-B ligand (RankL), Dickkopf WNT Signaling Pathway Inhibitor 1 (Dkk1), sclerostin (Sost), Cyclin D1 (Ccnd1), Axin2, and Ki67.

TABLE 4 Changes in gene expression in bone with Wnt surrogate molecule (1RC07-3) and romosozumab (Rxmab) Therapy in mice Fold Induction + /− SEM relative to Bgal mean value gene Time(hr) Bgal Rzmab 1RC073 RunX2 24 1.0 + /− 0.3  8.8 + /− 4.4   9.6 + /− 3.6**   48 1.0 + /− 0.7  1.9 + /− 1.0   1.5 + /− 0.5     ColA1 24 1.0 + /− 0.2  3.9 + /− 2.0   3.0 + /− 1.0     48 1.0 + /− 0.5  7.0 + /− 3.3   2.7 + /− 1.5     Dmp1 24 1.0 + /− 0.2  2.7 + /− 1.1   4.8 + /− 2.9     48 1.0 + /− 0.6  1.6 + /− 0.7   3.8 + /− 1.8     Alp 24 1.0 + /− 0.4 18.3 + /− 11.7 16.5 + /− 6.4*    48 1.0 + /− 0.6  4.0 + /− 1.8   5.0 + /− 1.9     RankL 24 1.0 + /− 0.3  8.6 + /− 4.5   8.7 + /− 3.3     48 1.0 + /− 0.7  1.5 + /− 0.6   4.3 + /− 1.4     Dkk1 24 1.0 + /− 0.2 10.5 + /− 5.1  17.9 + /− 6.0**   48 1.0 + /− 0.5  4.3 + /− 2.5   2.8 + /− 1.3     Sost 24 1.0 + /− 0.2  2.1 + /− 1.0   2.5 + /− 0.9     48 1.0 + /− 0.6  5.1 + /− 2.3   9.0 + /− 4.2**   Ccnd1 24 1.0 + /− 0.4  3.3 + /− 0.8   3.4 + /− 1.8     48 1.0 + /− 0.6  2.5 + /− 1.2   2.4 + /− 1.2     Axin2 24 1.0 + /− 0.5  3.2 + /− 1.1   6.1 + /− 1.8**   48 1.0 + /− 0.8  3.6 + /− 2.3   4.6 + /− 1.6*    Ki67 24 1.0 + /− 0.3  7.7 + /− 4.3  10.0 + /− 3.2     48 1.0 + /− 0.5  1.0 + /− 0.5   25.5 + /− 12.2 *** * p<0.05, ** p<0.005, 2-way ANOVA comparison with Bgal control *** p<0.001, 2-way ANOVA comparison with both Bgal and romosozumab

In comparison to anti-sclerostin antibody (romosozumab) treatment, gene expression signatures over the time points were distinct with Wnt surrogate molecule therapy, with induction of more robust Axin2 and Ki67 expression than that caused by romosozumab treatment.

Example 22 In Vivo Liver Regeneration Model and Characterization of AAV-Delivered Wnt Surrogates

In vivo experiments were conducted by infecting approximately 8-weeks old C57BL/6J mice with an AAV vector that expressed Flag- and His-tagged 18R5-DKK1c protein (AAV-18R5-DKK1c-FlagHis). 18R5-DKK1c is a fusion protein containing the frizzled binding antibody, 18R5, in scFv format fused to DKK1c, as described in PCT Publication WO2016/040895, e.g., FIG. 5 . Control mice were either injected sub-cutaneously with phosphate-buffered saline (PBS) only or romosozumab (10 mg/kg), or injected intravenously (IV) with an AAV vector that expressed green fluorescent protein (GFP) (AAV-CAG-GFP), or an AAV vector that expressed a fusion protein comprising an anti-GFP scFv fused to a mutant DKK1c (AAV-ScFv (anti-GFP)-DKK1cF234K-Flag-His). 28 days after infection, animals were weighed and sacrificed. The liver was weighed, and the liver to body weight ratio calculated. The content of the small intestine and colon was removed by flushing with phosphate-buffered saline and gentle pressure to expel the content. The small intestine and colon were then weighed.

Systemic expression of 18R5-DKK1c-FlagHis resulted in a significant liver weight increase (FIG. 37A). Systemic expression of the negative controls, eGFP or anti-eGFP-Dkk1cF234K, did not affect the liver to body weight ratio. Administration of the romosozumab recombinant protein or vehicle control, did not affect the liver to body weight ratio.

None of the treatments affected the small intestine (FIG. 37B) or colon (FIG. 37C) to body weight ratio.

These studies showed that 18R5-DKK1c-FlagHis increases liver weight, but not that of the small or large intestines. This suggests that 18R5-DKK1c-FlagHis can promote liver regeneration.

Example 23 In Vivo Liver Regeneration Model and Characterization of Recombinantly Produced Wnt Surrogates

In vivo experiments were conducted by treating mice with recombinantly produced anti-eGFP, R2M3-26, 1R-C07-26, romosozumab or Rspo2 proteins at various dosages. The Rspo2 protein is a fusion protein between a short splice variant of the Rspo2 gene and a human Fc fragment.

In one study, mice were housed 4 per cage and n=8 per treatment group were used. Approximately 8-weeks old C57BL6/J mice were administered recombinant proteins, anti-eGFP (1 mg/kg), R2M3-26 (1 or 10 mg/kg) or 1R-C07-26 (1, 5 or 10 mg/kg), twice weekly intra-peritoneally (i.p.) for four weeks. In addition, groups of mice were administered romosozumab (30 mg/kg) or PBS vehicle control, subcutaneously.

Mice were weighed at the beginning and throughout the treatment. None of the treatment with recombinant proteins affected total body weight significantly (FIG. 38A). On day 28, the liver was weighed, and the liver to body weight ratio calculated (FIG. 38B). The highest dose of R2M3-26 (10 mg/kg) resulted in a significant increase in liver to body weight ratio. None of the other treatment affected the liver weight significantly.

The increase in liver weight in response to R2M3-26 suggest that this recombinant protein can promote liver regeneration.

In another study, mice were housed 5 per cage and n=10 per treatment group were used. Approximately 8-weeks old C57BL/6J mice received a single i.p. injection containing anti-eGFP (0.56 mg/kg), R2M3-26 (0.3 mg/kg) or Rspo2 (0.46 mg/kg) alone, or with a combination of R2M3-26 (0.1 mg/kg) and Rspo2 (0.46 mg/kg).

24 or 48 hours after injection, mice were euthanized. A portion of the left liver lobe was snap-frozen in liquid nitrogen and stored at −80° C. for RNA analysis. Cyclin D1 and Ki67 expression were measured by performing qPCR using the Mm00432359_m1 Ccnd1 probe and the Mm01278617_m1 Ki67 probe (Thermofisher, 4331182). An additional portion of the left liver lobe was fixed in formalin and embedded in paraffin for immunohistochemistry analysis. Sections were stained with the anti-proliferating cell nuclear antigen (PCNA) (Abcam, ab18197) or anti-phospho-histone H3 (pH3) rabbit antibodies (Abcam, ab47297). The number of positive nuclei were counted using the image processing software, Image J.

Rspo2 alone increased Ki67 (FIG. 39A) and CyclinD1 (FIG. 39B) mRNA expression. In combination with R2M3-26, Rspo2 increased Ki67 and CyclinD1 expression further than with Rspo2 alone at 24 and 48 hours after treatment with recombinant proteins. Rspo2 alone increased the number of PCNA (FIG. 39C) and pH3 (FIG. 39D) positive nuclei in liver sections. In combination with R2M3-26, Rspo2 increased the number of PCNA and pH3 positive nuclei further than with Rspo2 alone at 48 hours after treatment with recombinant proteins.

These studies show that the proliferation markers, Ki67 mRNA, CyclinD1 mRNA and PCNA-positive nuclei, and the pH3 mitotic marker are induced by the R2M3-26 and Rspo2 recombinant proteins and suggest that these recombinant proteins can promote liver regeneration.

Example 24 In Vivo Chronic Liver Injury Model and Characterization of AAV-Delivered Wnt Surrogates

Two in vivo experiments were conducted in two, thioacetamide (TAA)- and CC14-induced, liver cirrhosis mouse models, thioacetamide (TAA)- and CC14-induced, to test the effect of AAV vectors expressing 18R5-DKK1c-FlagHis or Rspo2 protein on chronic liver injury. TAA was added to the drinking water of 6 weeks old C57BL/6J mice at a concentration of 300 mg/L throughout the TAA treatment duration. Mice were housed 5 per cage and groups of n=10 were used, except for the control groups without TAA treatment where n=5 per group was used.

In Study 1 (FIGS. 40A, 40C, 40E, 40G-H), mice with (n=10) or without (n=5) TAA treatment were weighed and sacrificed after 9 weeks of TAA addition into the drinking water, to measure baseline values. Livers were weighed and liver samples were collected for mRNA and histological analysis. TAA supplementation was maintained in the drinking water of the remaining mice and they were injected IV with AAV vectors that expressed an enhanced green fluorescent protein (eGFP) (3e10 genomic particles (GC)), 18R5-DKK1c-FlagHis (3e10 or 1e11 GC) or Rspo2 protein (1e11 GC) or a combination of 18R5-DKK1c-FlagHis (3e10 GC) and Rspo2 (1e11 GC). 5 age-matched naïve animals (no TAA) were kept as a negative control. Three weeks after AAV injection, all mice were weighed and euthanized. Livers were weighed and liver samples were collected for mRNA and histological analysis.

Treatment with 18R5Dkk1FH or Rspo2 resulted in a significant increase of liver weight (FIG. 40C) and liver to body weight ratio (FIG. 40E) in mice undergoing a continuous exposure to TAA. Treatment with a combination of 18R5Dkk1FH and Rspo2 resulted in a further increase in liver weight and liver to body weight ratio beyond that observed with either treatment alone. The combination 18R5Dkk1FH and Rspo2 treatment resulted in a decrease of the fibrosis marker Col1a1 mRNA expression (FIG. 40G). Histological liver sections were stained with Sirius red to visualize the accumulation of collagen in fibrotic area (FIG. 40H). Quantification of the percentage of red are, using Image J analysis software, showed a significant increase of fibrotic area in TAA-treated mice. A combination of 18R5Dkk1FH and Rspo2 resulted in a reversal of fibrotic area increase when compared to mice treated with the eGFP negative control. Treatment with Rspo2 alone also resulted in a significant but smaller reversal than the combination treatment.

In Study 2, (FIGS. 40B, 40D, 40F), mice were exposed to TAA-supplemented water for 11 weeks, and were returned to standard drinking thereafter, two days prior to AAV treatment. At the start of AAV treatment, mice exposed to TAA (n=10) or not (n=5) were weighed and sacrificed to collect liver samples for baseline measurements. The remaining mice were injected with AAV vectors that expressed an enhanced green fluorescent protein (eGFP) (1.3e11 genomic particles (GC)), 18R5-DKK1c-FlagHis (3e10 or 1e11 GC) or Rspo2 protein (1e11 GC) or a combination of 18R5-DKK1c-FlagHis (3e10 GC) and Rspo2 (1e11 GC). 5 age-matched naïve animals (no TAA) were kept as a negative control. Three weeks after AAV injection, all mice were weighed and euthanized. Livers were weighed and liver samples were collected for mRNA and histological analysis.

A similar liver weight and liver to body weight increase was observed in mice treated with 18R5-Dkk1c-FlagHis and Rspo2 either alone or in combination than what was observed in Study 1 (FIGS. 40D, 40F).

These studies show that 18R5-Dkk1c-FlagHis and Rspo2 can increase liver weight in a TAA-induced liver cirrhosis model and reduce fibrosis markers. These results suggest that 18R5-Dkk1c-FlagHis and Rspo2 can promote liver tissue repair after chronic liver injury.

Example 25 In Vivo Chronic Liver Injury Models and Characterization of Recombinantly Produced Wnt Surrogates

In vivo experiments were conducted in thioacetamide (TAA)-induced and CC14-induced mouse models of liver cirrhosis by treating mice with recombinantly produced anti-eGFP, R2M3-26 and Rspo2 proteins.

In the TAA-induced cirrhosis model, six weeks old male mice were exposed to TAA-supplemented drinking water (300 mg/L) for approximately 22 weeks (FIG. 41A). TAA exposure was removed two days prior to beginning of treatment with recombinant proteins and mice were provided with fresh drinking water. Mice were housed 5 per cage and n=10 per treatment group were used. In a mono treatment study (FIGS. 41B, 41D, 41F, 41H, 41J, 41L), mice were injected i.p. with anti-eGFP (1 mg/kg) or Rspo2 (1 mg/kg) twice weekly. In a combination treatment study (FIGS. 41C, 41E, 41G, 41I, 41K, 41M), mice were injected i.p. with anti-eGFP (1.3 mg/kg) or a combination of R2M3-26 (0.3 mg/kg) and Rspo2 (1 mg/kg) twice weekly. Mice were then weighed and sacrificed at day 3, 7 or 14 days after beginning of treatment. Groups of control mice without exposure to TAA (n=5 per group) were euthanized at day 0 and day 14 in both studies.

Treatment with Rspo2 protein alone or in combination with R2M3-26 resulted in an increase in liver to body weight ratio (FIGS. 41B and 41C) and a transient stimulation of the Wnt signaling pathway as shown by an increase in Axin2 expression (FIGS. 41D and 41E). Treatment with Rspo2 protein alone or in combination with R2M3-26 induced the following proliferation markers: cyclinD1 (FIGS. 41F, 41G) and Ki67 (FIGS. 41H and 41I) mRNA expression, PCNA (FIGS. 41J, 41K) and pH3 (FIGS. 41L, 41M) positive nuclei.

In an additional study, plasma was collected for prothrombin time measurement. Coagulation time is impaired in TAA-induced cirrhosis model as exemplified by an increase in pro-thrombin (PT) test to normal value ratio in mice exposed to TAA when compared to normal mice without TAA exposure (FIG. 41N). Treatment with Rspo2 (1 mg/kg) and R2M3-26 (0.3 mg/kg) reversed the prolongation in PT time as shown by the decrease in PT_(test)/PT_(normal) ratio at day 7 and day 14 after biweekly Rspo2 and R2M3-26 treatment.

These studies show that Rspo2 and R2M3-26 can stimulate liver cell proliferation and improve hepatocytes functional activity such as pro-thrombin time in a TAA-induced liver cirrhosis model. These results suggest that Rspo2 and R2M3-26 can promote liver tissue repair in chronic liver disease.

In the CC14-induced cirrhosis model, six-weeks old C57BL/6J male mice were injected i.p. with 2 ml/kg CC14 in mineral oil, twice weekly for 8 weeks (FIG. 42A). 3 days after the last CC14 injection, mice were injected i.p. twice weekly with the following recombinant proteins: anti-β-galactosidase (10 mg/kg), Rspo2 (1 or 10 mg/kg), or a combination of R2M3-26 (0.3 mg/kg) and Rspo2 (1 mg/kg). Three additional control groups were included: one group injected with CC14 but no proteins, one group injected with mineral oil, and one untreated age-matched naïve group. n=8 were used for each group. After two weeks of treatment with recombinant proteins, mice were weighed and sacrificed. Plasma was collected for pro-thrombin time measurement. Livers were weighed and liver samples were collected for histological analysis.

Treatment with R2M3-26 and Rspo2 resulted in a significant increase in liver to body weight ratio when compared to treatment with anti-β-galactosidase negative control (FIG. 42B). Treatment with Rspo2 (10 mg/kg) or a combination of R2M3-26 and Rspo2 resulted in a significant decrease in prothrombin time (FIG. 42C). Treatment with Rspo2 (10 mg/kg) or a combination of R2M3-26 and Rspo2 resulted in a significant reversal in fibrotic area, induced by CC14 (FIG. 42D).

This study showed that Rspo2 and R2M3-26 can induce an increase in liver weight, improve hepatocytes functional activity such as pro-thrombin time and reduce fibrosis markers in a CC14-induced liver cirrhosis model. These results suggest that Rspo2 and R2M3-26 can promote liver tissue repair in chronic liver disease.

Example 26 In Vivo Acute Liver Injury Model and Characterization of Recombinantly Produced Wnt Surrogates

In vivo experiments were conducted using acetaminophen-induced mouse models of acute liver injury by treating mice with recombinantly produced anti-eGFP, R2M3-26 and Rspo2 proteins.

Eight-week old C57BL/6 male mice were housed 5 per cage. n=10 were used per group. Mice were fasted overnight for 12 hours. Acetaminophen (APAP) was administered i.p. at a sublethal dose (300 mg/kg).

In a first study, anti-eGFP (0.3 mpk) or R2M3-26 (0.3 mpk) were injected i.p. either immediately after or 3 or 6 hours after APAP injection (FIG. 43 ). Serum samples were collected at 24 and 48 hours after APAP injection for ALT measurements. Mice were sacrificed 48 hours after APAP injection and liver samples were collected for mRNA analysis.

R2M3-26 treatment did not affect ALT level significantly (FIG. 43B). Treatment with R2M3-26 induced cyclinD1 (FIG. 43C) and Ki67 (FIG. 43D) mRNA significantly, beyond the level induced by APAP treatment alone.

In a second study, human Fc (0.46 mg/kg) or Rspo2 (0.46 mg/kg) were injected i.p. either immediately after or 3 or 6 hours after APAP injection (FIG. 44 ). Serum samples were collected at 24 and 48 hours after APAP injection. Liver samples were collected at 48 hours after APAP injection. Rspo2 treatment did not affect ALT serum level significantly (FIG. 44B). Treatment with Rspo2 induced cyclinD1 (FIG. 44C) and Ki67 (FIG. 43D) mRNA significantly, beyond the level induced by APAP treatment alone.

In a third study, anti-eGFP (0.56 mg/kg) or a combination of R2M3-26 (0.1 mg/kg) and Rspo2 (0.46 mg/kg) were injected i.p. 3 hours after APAP administration. Serum and liver samples were collected at 24, 36, 48 and 60 hours after APAP injection for ALT measurements and mRNA analysis.

R2M3-26 and Rspo2 combination treatment did not affect ALT level significantly (FIG. 45B). Treatment with R2M3-26 and Rspo2 induced cyclinD1 (FIG. 45C) and Ki67 (FIG. 45D) mRNA significantly, beyond the level induced by APAP treatment alone.

An additional study was performed to evaluate the effect of Rspo2 and R2M3-26 on the survival of mice treated with a dose of 600 mg/kg of acetaminophen (FIG. 46 ). Anti-eGFP (0.3 mg/kg), R2M3-26 (0.3 mg/kg), Rspo2 (0.46 mg/kg) or a combination of R2M3-26 (0.1 mg/kg) and Rspo2 (0.46 mg/kg) were injected i.p. 3 hours after APAP administration. Mice were monitored several times daily over the next 96 hours. A consistent trend in improving survival was observed in groups treated with R2M3-26 (FIG. 46B), Rspo2 (FIG. 46C) or a combination of R2M3-26 and Rspo2 (FIG. 46D).

These studies show that Rspo2 and R2M3-26 can induce proliferation markers beyond that induced spontaneously in APAP-induced acute injury model. These results suggest that Rspo2 and R2M3-26 can enhance liver tissue repair after an acute liver injury.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above-detailed description.

In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. 

What is claimed is:
 1. A soluble, multivalent, multispecific Wnt surrogate molecule, wherein the Wnt surrogate molecule comprises: (i) one or more Fzd binding regions that specifically binds to one or more Frizzled (Fzd) receptors, wherein the one or more Fzd binding regions comprise one or more antibody or antigen-binding fragment of the antibody, wherein the one or more antibody or the antigen-binding fragment comprises: a CDRH1 of SEQ ID NO: 307, a CDRH2 of SEQ ID NO: 501, a CDRH3 of SEQ ID NO: 964, a CDRL1 of SEQ ID NO: 1098, a CDRL2 of SEQ ID NO: 1175, and a CDRL3 of SEQ ID NO: 1372; and (ii) one or more LRP5/6 binding regions that specifically binds to a Low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) and/or a Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6), wherein the one or more LRP5/6 binding regions comprise one or more antibody or antigen-binding fragment of the antibody, wherein the one or more antibody or the antigen-binding fragment comprises: a CDRH1 of SEQ ID NO: 1618, a CDRH2 of SEQ ID NO: 1715, and a CDRH3 of SEQ ID NO:
 2012. 2. The Wnt surrogate molecule of claim 1, wherein the Wnt surrogate molecule comprises one or more polypeptides having at least 90% identity to the sequence set forth in SEQ ID NO: 137 and one or more polypeptide having at least 90% identity to the sequence set forth in SEQ ID NO:
 153. 3. The Wnt surrogate molecule of claim 1, wherein the Fzd binding region comprises a full immunoglobulin G (IgG), comprising two light chains and two heavy chains, and the LRP5/6 binding region comprises a VHH.
 4. The Wnt surrogate molecule of claim 3, wherein each of the one or more LRP5/6 binding regions is fused to the N-terminus of one of the light chains.
 5. A pharmaceutical composition comprising a physiologically acceptable excipient, diluent, or carrier, and the Wnt surrogate molecule according to claim
 1. 6. The Wnt Surrogate molecule of claim 4, wherein each of the one or more LRP5/6 binding regions is fused to one of the light chains via a linker moiety.
 7. The Wnt surrogate molecule of claim 6, wherein the Wnt surrogate molecule comprises: (i) two Fzd binding regions, wherein each of the Fzd binding regions comprise one or more antibody or antigen-binding fragment thereof, wherein the one or more antibody or the antigen-binding fragment thereof comprises: a CDRH1 of SEQ ID NO: 307, a CDRH2 of SEQ ID NO: 501, a CDRH3 of SEQ ID NO: 964, a CDRL1 of SEQ ID NO: 1098, a CDRL2 of SEQ ID NO: 1175, and a CDRL3 of SEQ ID NO: 1372; and (ii) two LRP5/6 binding regions, wherein each of the LRP5/6 binding regions comprise one or more antibody or antigen-binding fragment thereof, wherein the one or more antibody or the antigen-binding fragment thereof comprises: a CDRH1 of SEQ ID NO: 1618, a CDRH2 of SEQ ID NO: 1715, and a CDRH3 of SEQ ID NO:
 2012. 8. The Wnt surrogate molecule of claim 6, wherein the Wnt surrogate molecule comprises two polypeptides having at least 90% identity to the sequence set forth in SEQ ID NO: 137 and two polypeptides having at least 90% identity to the sequence set forth in SEQ ID NO:
 153. 9. A pharmaceutical composition comprising the physiologically acceptable excipient, diluent, or carrier, and the Wnt surrogate molecule of claim
 7. 10. A pharmaceutical composition comprising the physiologically acceptable excipient, diluent, or carrier, and the Wnt surrogate molecule of claim
 8. 